JP6161607B2 - How to determine the presence or absence of different aneuploidies in a sample - Google Patents

Description

  The present invention relates generally to the field of diagnostics and provides a method for determining variations (polymorphisms) in the total amount of nucleic acid sequences in a mixture of nucleic acids derived from different genomes. In particular, this method is applicable to non-invasive prenatal diagnosis and diagnosis and monitoring of metastatic progression in cancer patients.

  One of the major efforts in human medical research is the discovery of genetic abnormalities that cause adverse health effects. In many cases, specific genes and / or important diagnostic markers have been identified in portions where there are abnormal copy numbers of the genome. For example, in prenatal diagnosis, excess or missing copies across the chromosome are often appearing lesions. In cancer, copy deletions or duplications in whole chromosomes or chromosome fragments (segments) and high levels of amplification in specific regions of the genome appear in common.

  Much information on copy number variation (variation) has been obtained by cytogenetic elucidation that allows recognition of structural abnormalities. Common procedures for genetic screening and biological dosimetry have used invasive procedures, such as amniocentesis, to collect cells for karyotyping. Recognizing the need for faster tests that do not require cell culture, fluorescent in situ hybridization (FISH), quantitative fluorescent PCR (QF-PCR) and arrays Comparative genomic hybridization (CGH) has been developed as a molecular cytogenetic analysis method for analyzing copy number variation.

  The advent of technology that allows sequencing of the entire genome in a relatively short time, and circulating cell-free DNA (cfDNA), without the risks associated with invasive sampling methods, It has provided an opportunity to compare genetic material from one chromosome to be compared with chromosomes from other genetic material. However, the limitations of existing methods, including the limited sensitivity of stemming from a limited level of cfDNA and the sequencing bias of the technique of stemming due to the inherent nature of genomic information, Underlying the persistent desire for non-invasive methods that provide any or all of the availability for reliable copy number polymorphism diagnosis in various clinical contexts.

  The present invention fulfills some of the above needs and provides the advantage of providing a reliable method that can be applied, at least, to non-invasive prenatal diagnosis and to the diagnosis and monitoring of metastatic progression in cancer patients. .

  The present invention relates to a copy of a sequence of interest in a test sample having a nucleic acid mixture in which the total amount of one or more sequences of interest is known or concerned that there is a difference in the total amount of the sequences. A method for determining copy number variations (CNV) is provided. This method represents a statistical approach that is responsible for the accrual variation stemming process from interchromosomal and inter-sequence variation associated with the process. This method is applicable to determine CNVs in any fetal aneuploidy, and CNVs known or concerned about various medical disorders. CNVs that can be determined by the method of the present invention include trisomy and monosomy in any one or more of chromosomes 1-22, X and Y, other chromosomal polysomy, and one or more of the chromosomes. Fragment deletions and / or duplications are included, which can be determined by sequencing the nucleic acid of the test sample only once. Any aneuploidy can be determined from the sequence information obtained by sequencing the nucleic acid of the test sample only once.

  In one embodiment, the present invention provides a method for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids. The method of the present invention comprises (a) obtaining sequence information relating to fetal and maternal nucleic acids in the maternal test sample, and (b) using the sequence information from chromosomes 1 to 22, X chromosome and Y chromosome. Identifying the number of sequence tags for each of any four or more selected chromosomes of interest and identifying the number of sequence tags for each of the four or more selected chromosomes of interest of normalized chromosome sequences; (C) using the sequence tag number identified for each of the any four or more chromosomes of interest and the sequence tag number identified for the normalized chromosome sequence, Calculating a single chromosome dose for each of the four or more chromosomes of interest; and (d) the single stain of each of the four or more of the chromosomes of interest. Each somatic dose is compared to a threshold value for each of the any four or more chromosomes of interest, thereby determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in the maternal test sample. Determining. Step (a) sequences at least a portion of the nucleic acid molecules in the test sample to obtain sequence information regarding the fetal and maternal nucleic acid molecules in the test sample. In some embodiments, step (c) comprises the step of identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each of the chromosomes of interest, and the normalized chromosomal sequence of each of the chromosomes of interest. And calculating as a ratio to the number of sequence tags identified for. In some embodiments, step (c) comprises (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest. Calculating a sequence tag density ratio for each chromosome of interest; (ii) associating the number of sequence tags identified for the normalized chromosome sequence in step (b) with the length of each normalized chromosome Calculating the sequence tag density ratio of each normalized chromosome, and (iii) using the sequence tag density ratio calculated in steps (i) and (ii), respectively, Calculating the single-chromosome dose of the sequence tag density ratio of the chromosome of interest and the chromosome of interest respectively. Calculating as a ratio of the sequence tag density ratio in the normalized chromosomal sequence.

  In another embodiment, a method is provided for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids. The method of the present invention comprises (a) obtaining sequence information relating to fetal and maternal nucleic acids in the maternal test sample, and (b) using the sequence information from chromosomes 1 to 22, X chromosome and Y chromosome. Identifying the number of sequence tags for each of any four or more selected chromosomes of interest and identifying the number of sequence tags for each of the four or more selected chromosomes of interest of normalized chromosome sequences; (C) using the sequence tag number identified for each of the any four or more chromosomes of interest and the sequence tag number identified for the normalized chromosome sequence, Calculating a single chromosome dose for each of the four or more chromosomes of interest; and (d) the single stain of each of the four or more of the chromosomes of interest. Each somatic dose is compared to a threshold value for each of the any four or more chromosomes of interest, thereby determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in the maternal test sample. The arbitrary four or more chromosomes of interest selected from chromosomes 1-22, X, and Y were selected from chromosomes 1-22, X, and Y. At least 20 chromosomes shall be included and the presence or absence of at least 20 different complete fetal chromosomal aneuploidies is determined. The step (a) sequences at least a portion of the nucleic acid molecule in the test sample to obtain sequence information regarding the fetal and maternal nucleic acid molecules in the test sample. In some embodiments, the step (c) comprises the step of identifying a single chromosomal dose of each chromosome of interest, the number of sequence tags identified for each of the chromosomes of interest, and the normalized chromosome of each of the chromosomes of interest. Calculating as a ratio to the number of sequence tags identified for the sequence. In some other embodiments, step (c) comprises (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest. (Ii) calculating the sequence tag density ratio of each chromosome of interest by (ii) the number of the sequence tags identified for the normalized chromosome sequence in step (b) to the length of each normalized chromosome Calculating the sequence tag density ratio of each of the normalized chromosomes by associating, and (iii) using the sequence tag density ratio calculated in steps (i) and (ii), Calculating each single chromosomal dose, the chromosomal dose comprising the sequence tag density ratio in the chromosome of interest and each of the chromosomes of interest. Calculating as a ratio of the sequence tag density ratio in the normalized chromosomal sequence.

  In another embodiment, a method is provided for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids. The method of the present invention comprises (a) obtaining sequence information relating to fetal and maternal nucleic acids in the maternal test sample, and (b) using the sequence information from chromosomes 1 to 22, X chromosome and Y chromosome. Identifying the number of sequence tags for each of any four or more selected chromosomes of interest and identifying the number of sequence tags for each of the four or more selected chromosomes of interest of normalized chromosome sequences; (C) using the sequence tag number identified for each of the any four or more chromosomes of interest and the sequence tag number identified for the normalized chromosome sequence, Calculating a single chromosome dose for each of the four or more chromosomes of interest; and (d) the single stain of each of the four or more of the chromosomes of interest. Each somatic dose is compared to a threshold value for each of the any four or more chromosomes of interest, thereby determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in the maternal test sample. And any four or more chromosomes of interest selected from chromosomes 1-22, X and Y are all chromosomes 1-22, X and Y. The presence or absence of all different complete fetal chromosome aneuploidies of chromosomes 1-22, X and Y. The step (a) sequences at least a portion of the nucleic acid molecule in the test sample to obtain sequence information regarding the fetal and maternal nucleic acid molecules in the test sample. In some embodiments, the step (c) comprises the step of identifying a single chromosomal dose of each chromosome of interest, the number of sequence tags identified for each of the chromosomes of interest, and the normalized chromosome of each of the chromosomes of interest. Calculating as a ratio to the number of sequence tags identified for the sequence. In some other embodiments, step (c) associates the number of sequence tags identified for each of the chromosomes of interest in (i) step (b) with the length of each of the chromosomes of interest. Calculating a sequence tag density ratio for each of the chromosomes of interest, (ii) determining the number of the sequence tags identified for the normalized chromosome sequence in step (b) by the length of each normalized chromosome Calculating a sequence tag density ratio for each of the normalized chromosomes, and (iii) using the sequence tag density ratio calculated in steps (i) and (ii), Calculating a single chromosome dose for each chromosome, the chromosome dose comprising the sequence tag density ratio in the chromosome of interest and the chromosome of interest Calculating as a ratio of each normalized chromosomal sequence to the sequence tag density ratio.

  In any of the above-described embodiments, the normalized chromosome sequence is a single chromosome selected from chromosomes 1 to 22, the X chromosome, and the Y chromosome. As an alternative, the normalized chromosomal sequence is a chromosome group selected from chromosomes 1-22 and X and Y chromosomes.

  In another embodiment, a method is provided for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids. The method comprises (a) obtaining sequence information relating to fetal and maternal nucleic acids in the maternal test sample, and (b) selecting from chromosomes 1-22, X chromosome and Y chromosome using the sequence information. Identifying the number of sequence tags for each of any one or more of the chromosomes of interest and identifying the number of sequence tags of each of the normalized fragment sequences of each of the one or more of the chromosomes of interest; (c) using the number of sequence tags identified for each of the one or more chromosomes of interest and the number of sequence tags identified for the normalized fragment sequence, Calculating a single chromosomal dose for each of the one or more chromosomes of interest; and (d) the single chromosomal dose of each of the any one or more chromosomes of interest. Is compared to the threshold of each of the one or more chromosomes of interest to determine the presence or absence of any one or more different complete fetal chromosomal aneuploidies in the maternal test sample. A step of performing. The step (a) sequences at least a portion of the nucleic acid molecule in the test sample to obtain sequence information regarding the fetal and maternal nucleic acid molecules in the test sample. In some embodiments, step (c) comprises the step of identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each of the chromosomes of interest, and the normalized fragment of each of the chromosomes of interest. Calculating as a ratio to the number of sequence tags identified for the sequence. The step (c) includes (i) associating the number of the sequence tags identified for each of the chromosomes of interest in the step (b) with the length of each of the chromosomes of interest, thereby Calculating the sequence tag density ratio of: (ii) correlating the number of sequence tags identified for the normalized fragment sequence in step (b) with the length of each normalized chromosome, Calculating a sequence tag density ratio for each of the fragment fragments, and (iii) using the sequence tag density ratio calculated in steps (i) and (ii) to calculate a single chromosome dose for each of the chromosomes of interest. And calculating the chromosome dose in the sequence tag density ratio in the chromosome of interest and the normalized fragment sequence of each of the chromosomes of interest. And calculating the ratio to the sequence tag density ratio.

  In another embodiment, a method is provided for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids. The method comprises (a) obtaining sequence information relating to fetal and maternal nucleic acids in the maternal test sample, and (b) selecting from chromosomes 1-22, X chromosome and Y chromosome using the sequence information. Identifying the number of sequence tags for each of any one or more of the chromosomes of interest and identifying the number of sequence tags of each of the normalized fragment sequences of each of the one or more of the chromosomes of interest; (c) using the number of sequence tags identified for each of the one or more chromosomes of interest and the number of sequence tags identified for the normalized fragment sequence, Calculating a single chromosomal dose for each of the one or more chromosomes of interest; and (d) the single chromosomal dose of each of the any one or more chromosomes of interest. Is compared to the threshold of each of the one or more chromosomes of interest to determine the presence or absence of any one or more different complete fetal chromosomal aneuploidies in the maternal test sample. Any one or more chromosomes of interest selected from Chromosomes 1-22, X and Y are at least 20 selected from Chromosomes 1-22, X and Y The presence or absence of at least 20 different complete fetal chromosome aneuploidies is determined. The step (a) sequences at least a portion of the nucleic acid molecule in the test sample to obtain sequence information regarding the fetal and maternal nucleic acid molecules in the test sample. In some embodiments, step (c) comprises the step of identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each of the chromosomes of interest, and the normalized fragment of each of the chromosomes of interest. Calculating as a ratio to the number of sequence tags identified for the sequence. In some other embodiments, step (c) associates the number of sequence tags identified for each of the chromosomes of interest in (i) step (b) with the length of each of the chromosomes of interest. Calculating the sequence tag density ratio of each of the chromosomes of interest, (ii) the number of the sequence tags identified for the normalized fragment sequence in step (b) is the length of each normalized chromosome (Iii) calculating the sequence tag density ratio of each of the normalized fragment sequences, and (iii) using the sequence tag density ratio calculated in steps (i) and (ii), Calculating a single chromosomal dose for each of the target chromosomes, the chromosomal dose comprising the sequence tag density ratio in the chromosome of interest and the chromosome of interest; Calculating as a ratio of each normalized fragment sequence to the sequence tag density ratio.

  In another embodiment, a method is provided for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids. The method comprises (a) obtaining sequence information relating to fetal and maternal nucleic acids in the maternal test sample, and (b) selecting from chromosomes 1-22, X chromosome and Y chromosome using the sequence information. Identifying the number of sequence tags for each of any one or more of the chromosomes of interest and identifying the number of sequence tags of each of the normalized fragment sequences of each of the one or more of the chromosomes of interest; (c) using the number of sequence tags identified for each of the one or more chromosomes of interest and the number of sequence tags identified for the normalized fragment sequence, Calculating a single chromosomal dose for each of the one or more chromosomes of interest; and (d) the single chromosomal dose of each of the any one or more chromosomes of interest. Is compared to the threshold of each of the one or more chromosomes of interest to determine the presence or absence of any one or more different complete fetal chromosomal aneuploidies in the maternal test sample. And any one or more chromosomes of interest selected from chromosomes 1-22, X and Y are all chromosomes 1-22, X and Y, Determine the presence or absence of all different complete fetal chromosomal aneuploidies of chromosomes 1-22, X and Y. The step (a) sequences at least a portion of the nucleic acid molecule in the test sample to obtain sequence information regarding the fetal and maternal nucleic acid molecules in the test sample. In some embodiments, step (c) comprises the step of identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each of the chromosomes of interest, and the normalized fragment of each of the chromosomes of interest. Calculating as a ratio to the number of sequence tags identified for the sequence. In some other embodiments, step (c) associates the number of sequence tags identified for each of the chromosomes of interest in (i) step (b) with the length of each of the chromosomes of interest. Calculating the sequence tag density ratio of each of the chromosomes of interest, (ii) the number of the sequence tags identified for the normalized fragment sequence in step (b) is the length of each normalized chromosome (Iii) calculating the sequence tag density ratio of each of the normalized fragment sequences, and (iii) using the sequence tag density ratio calculated in steps (i) and (ii), Calculating a single chromosomal dose for each of the target chromosomes, the chromosomal dose comprising the sequence tag density ratio in the chromosome of interest and the chromosome of interest; Calculating as a ratio of each normalized fragment sequence to the sequence tag density ratio. In any one of the above embodiments, the different complete fetal chromosomal aneuploidy is selected from complete chromosomal trisomy, complete chromosomal monosomy, and complete chromosomal polysomy. For example, the different complete fetal chromosomal aneuploidies are trisomy 2, trisomy 8, trisomy 9, trisomy 21, trisomy 13, trisomy 16, trisomy 18, trisomy 22, 47, XXY, 47 , XXX, 47, XYY, and X monosomy.

In any one of the embodiments described above, steps (a)-(d) are repeated for test samples from different maternal specimens, and the method comprises any four or more in each of the test samples Determine the presence or absence of different complete fetal chromosomal aneuploidies. In any one of the above-described embodiments, the method of the present invention further includes an NCV calculation step of calculating a normalized chromosome value (NCV), wherein the NCV is expressed by the following equation: Let the dose relate to the average of the corresponding chromosomal doses in the eligible sample set,
here,
Are the estimated mean and standard deviation for chromosome jose in the eligible sample set, respectively, and x _ij is the observed NC chromosome dose in test sample i.

  In another embodiment, a method is provided for determining the presence or absence of different partial fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids. The method comprises (a) obtaining sequence information relating to fetal and maternal nucleic acids in the maternal test sample, and (b) selecting from chromosomes 1-22, X chromosome and Y chromosome using the sequence information. Identifying the number of sequence tags for each of any one or more fragments in any one or more of the chromosomes of interest, and any one or more of said one or more of the chromosomes of interest Identifying the number of sequence tags of the normalized fragment sequence of each of the further fragments, and (c) identifying for each of any one or more fragments in the any one or more chromosomes of interest Using the sequence tag number and the sequence tag number identified for the normalized fragment sequence, any one in the one or more chromosomes of interest. Calculating a single chromosome dose for each of the one or more fragments, and (d) each of the single fragment doses for each of any one or more fragments in any one or more of the chromosomes of interest. Comparing any one or more chromosomal fragments of each of the one or more chromosomes of interest to a threshold, thereby providing any one or more different partials in the maternal test sample. Determining the presence or absence of fetal chromosomal aneuploidy. The step (a) sequences at least a portion of the nucleic acid molecule in the test sample to obtain sequence information regarding the fetal and maternal nucleic acid molecules in the test sample.

In another embodiment, the step (c) comprises the step of dividing a single fragment dose of each of any one or more chromosome fragments in the chromosome of interest into any one or more chromosome fragments in the chromosome of interest. Calculating as a ratio between the number of sequence tags identified for each and the number of sequence tags identified for each normalized fragment sequence of any one or more chromosome fragments in the chromosome of interest Have In some embodiments, step (c) comprises (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest. Calculating a sequence tag density ratio for each chromosome of interest; (ii) associating the number of sequence tags identified for the normalized fragment sequence in step (b) with the length of each normalized chromosome Calculating the sequence tag density ratio of each of the normalized fragment sequences, and (iii) using the sequence tag density ratio calculated in steps (i) and (ii), Calculating each single chromosomal dose, the chromosomal dose comprising the sequence tag density ratio in the chromosome of interest and each of the chromosomes of interest. Calculating the ratio of the normalized tag sequence to the sequence tag density ratio. The method further includes an NSV calculation step of calculating a normalized segment value (NSV), wherein the NSV determines the fragment dose as a corresponding fragment dose in an eligible sample set as follows: The value associated with the average of
here,
Are the estimated mean and standard deviation for the jth fragment dose in the qualifying sample set, respectively, and x _ij is the observed jth fragment dose in test sample i.

  Chromosome dose or fragment dose is determined using a normalized fragment sequence In the above-described method embodiment, the normalized fragment sequence is any one or more of chromosomes 1-22 and X and Y. It is set as the single fragment | piece in the above. As an alternative, the normalized fragment sequence is a fragment group in any one or more of chromosomes 1-22 and X and Y.

  Steps (a)-(d) of the method for determining the presence or absence of partial fetal chromosomal aneuploidy are repeated for test samples from different maternal specimens. Partial fetal chromosomal aneuploidy that can be determined by the method of the present invention includes partial aneuploidy of any fragment in any chromosome. Partial fetal chromosomal aneuploidy can be selected from partial duplication, partial growth, partial insertion and partial deletion. Examples of partial aneuploidy that can be determined by the method of the present invention include partial monosomy of chromosome 1, partial monosomy of chromosome 4, partial monosomy of chromosome 5, partial monosomy of chromosome 7, partial monosomy of chromosome 11, There is a partial monosomy of chromosome 15, a partial monosomy of chromosome 17, a partial monosomy of chromosome 18, and a partial monosomy of chromosome 22.

  In any one of the embodiments described above, the test sample is a maternal sample selected from blood, plasma, serum, urine and saliva samples. In any one embodiment, the test sample can be a plasma sample. The nucleic acid molecule of the maternal sample is a mixture of fetal and maternal cell-free DNA molecules. Nucleic acid sequencing can be performed using next generation sequencing (NGS). In some embodiments, the sequencing uses sequencing by synthesis with a reversible dye terminator. In another embodiment, the sequencing is sequencing by ligation. In yet another embodiment, the sequencing is a single molecule sequencing. Optionally, the amplification step shall be performed prior to sequencing.

  In another embodiment, the present invention provides a method for determining the presence or absence of any 20 or more different complete fetal chromosomal aneuploidies in a maternal plasma test sample comprising a mixture of fetal and maternal cell-free DNA molecules. To do. The method comprises (a) sequencing at least a portion of a cell-free DNA molecule to obtain sequence information regarding fetal and maternal cell-free DNA molecules in the maternal plasma test sample; and (b) the sequence information. To identify the number of sequence tags for each of any 20 or more chromosomes of interest selected from chromosomes 1-22 and X and Y chromosomes, and any 20 or more of the chromosomes of interest Identifying the number of sequence tags for each normalized chromosome, and (c) identifying the number of sequence tags identified for each of the 20 or more of the chromosomes of interest, and identifying the normalized chromosomes Calculating a single chromosomal dose for each of the any 20 or more chromosomes of interest using the sequence tag number; and ( d) comparing each single chromosomal dose of each of the any 20 or more chromosomes of interest to a threshold of each of the any of the 20 or more chromosomes of interest, thereby any of the maternal plasma test samples; Determining the presence or absence of 20 or more different complete fetal chromosomal aneuploidies.

  In another embodiment, the present invention provides a method of identifying a sequence of interest in a test sample, ie, a copy number variation (variation) of a clinically relevant sequence, the method of the present invention comprising: A sample collection step for collecting a test sample and a plurality of eligible samples, wherein the test sample comprises a test nucleic acid molecule and the plurality of eligible samples comprise a qualified nucleic acid molecule; (b ) Obtaining sequence information regarding fetal and maternal nucleic acids in the test sample; and (c) qualifying the qualifying sequence dose of the qualifying sequence of interest in the plurality of qualifying samples based on sequencing of qualifying nucleic acid molecules. A sequence dose calculation step, wherein the calculation of a qualified sequence dose comprises at least one qualified positive for the sequence of qualified interest. Determining a parameter in the normalized sequence, the qualified sequence dose calculation step, and (d) a qualified normalized sequence identification step that identifies at least one qualified normalized sequence based on the qualified sequence dose, comprising: A qualified normalized sequence identification step, wherein said qualified normalized sequence identification step has minimum variability and / or maximum discriminability with respect to sequence dose in said plurality of qualified samples; and (e) in said test sample A test sequence dose calculation step for calculating a test sequence dose for a test sequence of interest based on a sequence of nucleic acid molecules, the calculation of the test sequence dose in at least one normalized test sequence for the test sequence of interest Determining parameters, and at least one normalized test sequence is at least The test sequence dose calculation step corresponding to one eligible normalized sequence, (f) comparing the test sequence dose to at least one threshold, and (g) based on the results in step (f). Evaluating a copy number variation of the sequence of interest in the test sample. In one embodiment, the qualified sequence of interest and at least one qualified normalized sequence parameter relate the number of sequence tags mapped to the qualified sequence of interest to the number of tags mapped to the qualified normalized sequence, and the test sequence of interest and The parameter of at least one normalized test sequence shall relate the number of sequence tags mapped to the test sequence of interest to the number of tags mapped to the normalized test sequence. In some embodiments, step (b) is a step of sequencing at least a portion of the qualified nucleic acid molecule and the test nucleic acid molecule, whereby the sequencing is for the test sequence of interest and the qualified sequence, and at least Obtaining a plurality of mapped sequence tags for one test normalization sequence and at least one qualified normalization sequence, sequencing at least a portion of the nucleic acid molecules of the test sample, and fetus and mother in the test sample The step of obtaining the sequence information of the nucleic acid molecule. In some embodiments, the sequencing step is a next generation sequencing. In some embodiments, the sequencing method is massively parallel sequencing using sequencing by synthesis with a reversible dye terminator. In another embodiment, the sequencing method is sequencing by ligation. In some embodiments, sequencing shall include amplification. In other embodiments, the sequencing is a single molecule sequencing. The CNV of the sequence of interest is chromosomal or partial aneuploid. In some embodiments, the chromosomal aneuploidy is trisomy 2, trisomy 8, trisomy 9, trisomy 16, trisomy 21, trisomy 13, trisomy 18, trisomy 18, 47, XXY, 47, Select from XXX, 47, XYY, and X monosomy. In other embodiments, the partial aneuploidy is a partial chromosomal deletion or partial chromosomal insertion. In some embodiments, the CNV identified by the method of the invention is a chromosomal aneuploidy or partial aneuploidy associated with cancer. In some embodiments, the test sample and eligible sample are biological fluid samples, eg, plasma samples, taken from a pregnant specimen, such as a pregnant human specimen. In other embodiments, test biological fluid samples and qualified biological fluid samples, eg, plasma samples, are collected from specimens that are known or suspected of having cancer.

  Although the examples herein relate to humans and the terminology primarily relates to humans, the concepts of the present invention can be applied to the genome of any plant or animal.

INCORPORATION BY REFERENCE All patents, patent applications, and other documents, including any sequences described in the references cited herein, are specifically and individually listed by reference for each individual publication, patent or patent application. It is to be clearly incorporated herein as much as described. The relevant parts of all cited documents are incorporated herein by reference. However, citation of any document should not be construed as an admission that it is prior art of the present invention.

  The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized.

2 is a flowchart of a method 100 for determining the presence or absence of copy number polymorphism in a test sample having a nucleic acid mixture. Distribution of chromosomal doses for chromosome 21 determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human male or female fetus, A test sample of No. 21 trisomy is shown with (Δ) attached to the chromosomes 1 to 12 and the X chromosome. Distribution of chromosomal dose (chromosome) for chromosome 21 determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, A test sample of No. 21 trisomy is shown with (Δ) attached to chromosomes 1 to 22 and X chromosome, which is qualified as chromosome 21 (ie, normal). A distribution of chromosomal doses for chromosome 18 determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, A test sample of No. 18 trisomy is shown with (Δ) attached to the chromosomes 1 to 12 and the X chromosome. A distribution of chromosomal doses for chromosome 18 determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, A test sample of trisomy 18 is shown with (Δ) attached to chromosomes 1 to 22 and X chromosome (◯) with respect to normal that is qualified as chromosome 18, ie, normal. Distribution of chromosomal dose (chromosome) for chromosome 13 determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, A test sample of No. 13 trisomy is shown with (Δ) attached to the chromosomes 1 to 12 and the X chromosome that are qualified as chromosome 13 (ie, normal). Distribution of chromosomal dose (chromosome) for chromosome 13 determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, A test sample of No. 13 trisomy is shown with (Δ) attached to chromosomes 1 to 22 and X chromosome, which is qualified as chromosome 13 (ie, normal). Distribution of chromosomal doses (quantities) on the X chromosome determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, (46, XY; (◯)) is attached to the X chromosome dose of the child, (46, XX; (△)) is attached to the X chromosome dose of the girl, and (45, X; (+)) is attached to the X monosomy. A complex karyotype sample is shown with (Cplx (X)) attached to chromosomes 1 to 12. A distribution of chromosomal doses (quantities) for the X chromosome determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, (46, XY; (◯)) is attached to the X chromosome dose of the child, (46, XX; (△)) is attached to the X chromosome dose of the girl, and (45, X; (+)) is attached to the X monosomy. A complex karyotype sample is shown with (Cplx (X)) attached to chromosomes 1 to 22. A distribution of chromosomal doses (quantity) for the Y chromosome determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, (46, XY; (△)) is attached to the Y-chromosome dose of the child, (46, XX; (◯)) is attached to the X-chromosome dose of the girl, and (45, X; (+)) is attached to the X monosomy. A complex karyotype sample is shown with (Cplx (X)) attached to chromosomes 1 to 12. A distribution of chromosomal doses (quantity) for the Y chromosome determined by sequencing cfDNA extracted from a set of 48 blood samples taken from each subject pregnant with a human boy or girl fetus, (46, XY; (△)) is attached to the Y-chromosome dose of the child, (46, XX; (◯)) is attached to the X-chromosome dose of the girl, and (45, X; (+)) is attached to the X monosomy. A complex karyotype sample is shown with (Cplx (X)) attached to chromosomes 1 to 22. The coefficient of variation (CV) of chromosome 21 (black square), chromosome 18 (●) and chromosome 13 (black triangle) determined from the doses shown in FIGS. The coefficient of variation (CV) of the X chromosome (black square) and Y chromosome (●) determined from the doses shown in FIGS. 5 and 6, respectively, is shown. It is the cumulative distribution of the GC fraction by human chromosomes, the vertical axis represents the frequency of the chromosome, and the GC content is shown on the horizontal axis below the value. Fragment of chromosome 11 (81000082-10300013bp) determined by sequencing cfDNA extracted from a set of 7 eligible samples (◯) and 1 test sample (◇) collected from a pregnant human subject A sample from a subject bearing a fetus having a partial aneuploidy on chromosome 11 was identified. The distribution of the normalized chromosomal dose of chromosome 21 with respect to the standard deviation of the mean (Y axis) for the corresponding chromosome in a sample with no abnormality is shown. The distribution of the normalized chromosomal dose of chromosome 18 with respect to the standard deviation of the mean (Y axis) for the corresponding chromosome in a sample with no abnormality is shown. The distribution of the normalized chromosomal dose of chromosome 13 with respect to the standard deviation of the mean (Y axis) for the corresponding chromosome in a sample with no abnormality is shown. FIG. 5 shows the normalized chromosome dose distribution of the X chromosome relative to the standard deviation of the mean (Y axis) for the corresponding chromosome in a sample with no abnormalities. FIG. 6 shows the normalized chromosomal dose distribution of the Y chromosome relative to the standard deviation of the mean (Y axis) for the corresponding chromosome in a sample with no abnormalities. Using normalized chromosomes described in Example 6, the normalized chromosome values of chromosome 21 (◯), chromosome 18 (Δ), and chromosome 13 (□) determined in the sample from training set 1 are shown. . Using normalized chromosomes described in Example 6, the normalized chromosome values of chromosome 21 (◯), chromosome 18 (Δ), and chromosome 13 (□) determined in the sample from test set 1 are shown. . Tested using the normalization method by Chu et al. (Normalized for the number of sequence tags identified for the chromosome of interest with base sequence tag number obtained for the remaining chromosome in the sample; see Example 7) The normalized chromosome values of chromosome 21 (◯), chromosome 18 (Δ), and chromosome 13 (□) determined with the sample from set 1 are shown. Using normalized chromosomes systematically determined (as described in Example 7), chromosome 21 (◯), chromosome 18 (Δ), chromosome 13 (in the sample from training set 1) □) shows the normalized chromosome value. Using normalized chromosomes systematically determined (as described in Example 7), chromosomes 21 (◯), 18 (Δ), 13 (determined in samples from test set 1 ( □) shows the normalized chromosome value. The normalized chromosome values of chromosome 9 (O) determined in the sample from Test Set 1 are shown using systematically determined normalized chromosomes (as described in Example 7). The normalized chromosome values of the X chromosome (X axis) and the Y chromosome (Y axis) are shown, and as described in Example 7, five X monosomy samples identified in each of the training set and the test set are indicated by arrows. Show. The normalized chromosome values of the X chromosome (X axis) and the Y chromosome (Y axis) are shown, and as described in Example 7, three X monosomy samples identified in each of the training set and the test set are indicated by arrows. Show. The normalized chromosome values of chromosomes 1-22 determined in samples from test set 1 are shown using systematically determined normalized chromosomes (as described in Example 7).

  A copy number variation of the sequence of interest in a test sample having a nucleic acid mixture in which the total amount of one or more sequences of interest is known or that the total amount of the sequences is of concern A method for determining CNV: copy number variations is provided. Sequences of interest include genomic sequences ranging from kilobases (kb) to megabases (Mb) with respect to the entire chromosome known or concerned to be associated with a genetic condition or disease state. Examples of sequences of interest include the well-known aneuploidy, eg, chromosomes associated with trisomy 21 and chromosome fragments that are propagated in diseases such as cancer, eg, acute myeloid leukemia There is a chromosome associated with partial trisomy 8. CNVs that can be determined according to the present invention include monosomy and trisomy on any one or more of chromosomes 1-22, X and Y sex chromosomes, eg 45, X, 47, XXX, 47 , XXY, and 47, XYY, other chromosomal polysomy, ie, tetrasomy and pentasomy, including but not limited to XXXX, XXXXXX, XXXXY, XYYYY, deletion of segments (segments) in any one or more of the chromosomes and There is an overlap.

  The method of the present invention provides a statistical approach that is responsible for the accrual variability stemming process from inter-chromosome (intra-run) and inter-sequence (inter-run) variability associated with processing. This method is applicable to determine CNV in any fetal aneuploidy, and CNVs known or concerned about various medical disorders.

  Unless otherwise indicated, the practice of the present invention is common in the fields of molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing (sequencing) and recombinant DNA within the prior art. Including ordinary technology used. Such techniques are known to those skilled in the art and many documents and references (eg, Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Third Edition (Cold Spring Harbor), [2001]); See Ausubel et al., “Current Protocols in Molecular Biology” [1987]. )It is described in.

  Many ranges are included in the number defining the range. Any maximum numerical limitation set forth herein is intended to include any lower numerical limitation as such lower numerical limitation is expressly set forth herein. Any minimum numerical limitation set forth herein is intended to include any higher numerical limitation as such higher numerical limitation is expressly set forth herein. Every numerical range recited herein includes any narrow numerical range falling within such wider numerical range, as such narrower numerical range is expressly set forth herein. Intended.

  The headings provided herein are not intended to limit the various aspects or embodiments of the invention that can be understood by reference to the specification as a whole. Accordingly, as described above, the terms defined below are more fully defined by reference to the entire specification.

  Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Various scientific dictionaries containing the terms contained herein are known and available to those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some suitable methods and materials are described. Accordingly, the terms defined below are more fully described by reference to the entire specification. The present invention is not limited to the particular methodologies, procedures, and reagents described herein and may vary depending on the context used by those skilled in the art.

Definitions As used herein, the singular forms “a”, “an”, and “the” are intended to include plural references unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5 'to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

  As used herein, the term “assessing” refers to a chromosome by one of three types of determinations: “normal”, “affected”, and “no-call”. Means characterizing the aneuploidy state. For example, in the case of trisomy presence / absence determination, the determination of “normal” is determined by a parameter, for example, a value of a test chromosome dose that is less than a user-defined reliability threshold, and the determination of “abnormality” is a parameter, for example, a user-defined reliability The determination of “no call” is determined by a test chromosome dose that is between user-defined confidence thresholds that make a determination of a parameter, eg, “normal” or “abnormal”.

  As used herein, the term “copy number polymorphism” means a variation in copy number in a nucleic acid sequence having a length of 1 kb or more present in a test sample compared to the copy number of a nucleic acid sequence present in a qualified sample. A “copy number variation” is a sequence having a length of 1 kb or more in a nucleic acid, and the copy number difference compares a sequence of interest in a test sample with its sequence of interest in a qualified sample. To find out. Copy number polymorphisms include deletions including microdeletions, insertions including microinsertions, duplication, growth, inversion, translocation, and complex multisite mutations. CNV includes chromosomal aneuploidy and partial aneuploidy.

  As used herein, the term “aneuploidy” means an imbalance of genetic material caused by a deficiency or excess in an entire chromosome or a portion of a chromosome.

  As used herein, the terms “chromosomal aneuploidy” and “complete chromosomal aneuploidy” mean an imbalance of genetic material caused by a deficiency or excess in the entire chromosome, and germline aneuploidy and mosaic aneuploidy including.

  As used herein, the terms “partial aneuploidy” and “partial chromosomal aneuploidy” refer to an imbalance in genetic material caused by a deficiency or excess in part of a chromosome, for example, partial monosomy and partial trisomy, Including imbalances caused by deletions and insertions.

  As used herein, the term “aneuploid sample” means a sample that represents an analyte whose chromosome content is not euploid, that is, a sample that represents an analyte with an abnormal chromosome copy number.

  As used herein, the term “aneuploid chromosome” means a chromosome that is known or determined to be present in a sample of an abnormal copy number.

As used herein, the term “plurality” refers to a large number of nucleic acid molecules or nucleotide sequence tags (eg, chromosomal dose tags) sufficient to distinguish large differences in copy number variations in test and qualified samples used in the methods of the invention. ). In some embodiments, at least about 3 × 10 ⁶ base sequence tags, at least about 5 × 10 ⁶ base sequence tags, having at least about 40 × 40 base pair (bp) reads, at least about 8 × 10 ⁶ base sequence tags, at least about 10 × 10 ⁶ base sequence tags, at least about 15 × 10 ⁶ base sequence tags, at least about 20 × 10 ⁶ base sequence tags, at least about 30 × 10 ^Six base sequence tags, at least about 40 × 10 ⁶ base sequence tags, at least about 50 × 10 ⁶ base sequence tags are obtained from each test sample.

  As used herein, the terms “polynucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably and refer to covalent sequences of nucleotides (ie, RNA ribonucleotides and DNA deoxyribonucleotides). The 3 'position of the pentose in one nucleotide is linked by a phosphodiester group to the 5' position of the pentose in the next nucleotide, including sequences of any nucleic acid format, including but not limited to RNA, DNA, and cfDNA molecules. The term “polynucleotide” includes, but is not limited to, single and double helix polynucleotides.

  As used herein, the term “portion” means that the total amount of sequence information of fetal and maternal nucleic acid molecules in a biological sample is less than the sequence information of one human genome.

  As used herein, the term “test sample” refers to a sample comprising a mixture of nucleic acids having at least one nucleic acid sequence that is of concern for copy number variation. Nucleic acids present in a test sample are referred to as “test nucleic acids”.

  As used herein, the term “eligible sample” refers to a sample having a mixture of nucleic acids that is known and present in copy numbers compared to nucleic acids in a test sample, that is, a sample that is normal, ie, the sequence of interest has no aneuploidy. Yes, for example, a qualified sample used to identify a normalized chromosome of chromosome 21 is a sample that is not a trisomy sample.

  As used herein, the term “training set” means a sample set that can include samples with and without anomalies. Non-aberrant samples in the training set are used as qualifying samples to identify normalized sequences, e.g., normalized chromosomes, and the chromosomal doses of invariant samples are used to determine the threshold for each sequence of interest, e.g., each chromosome. Set. Samples with anomalies in the training set can be used to verify that test samples with anomalies can be easily distinguished from samples without anomalies.

  As used herein, the term “qualifying nucleic acid” can be used interchangeably with “qualifying sequence”, which is a test sequence or a sequence that is compared to the total amount of test nucleic acid. A qualified sequence is a known representation, ie, the total amount of qualified sequence is known and present in a biological sample. A “qualifying sequence of interest” is a qualifying sequence whose total amount is known in a qualifying sample, and is a sequence associated with a difference in sequence expression in an individual with a medical disorder.

  As used herein, the term “sequence of interest” refers to a nucleic acid sequence that is associated with a difference in sequence representation in a healthy individual versus a diseased individual. A sequence of interest is a misrepresentation in a medical or genetic disorder, ie, a sequence in a chromosome that is over- or under-represented. The sequence of interest can also be part of a chromosome, ie a chromosomal fragment (segment), or a chromosome. For example, the sequence of interest can be a gene that encodes a chromosome that is overexpressed in aneuploidy or a tumor suppressor that is underexpressed in cancer. Sequences of interest include sequences that are over- or under-represented in the entire or subpopulation of cells in the specimen. A “qualifying sequence of interest” is a sequence of interest in a qualified sample. A “test sequence of interest” is a sequence of interest in a test sample.

  As used herein, the term “normalized sequence” refers to a sequence that exhibits variation (polymorphism) in the number of base sequence tags (sometimes abbreviated as “sequence tags”) mapped between samples, A sequencing run that most closely resembles that of the sequence of interest used as and can most distinguish the anomalous samples from one or more anomalous samples. A “normalized chromosome” or “normalized chromosome sequence” is an example of a “normalized sequence”. A “normalized chromosomal sequence” can be composed of a single chromosome or a group of chromosomes. “Normalized fragment” is another example of “normalized sequence”. A “normalized fragment sequence” can be composed of a single fragment of a chromosome, or it can be composed of two or more fragments on the same or different chromosomes.

  As used herein, the term “discriminability” means a normalized chromosomal feature that can distinguish one or more abnormalities, ie, normal samples from one or more abnormalities, ie, aneuploid samples. To do.

  As used herein, the term “sequence dose” refers to a parameter that relates the sequence tag density of a sequence of interest to the tag density of a normalized sequence. “Test sequence dose” is a parameter that relates the sequence tag density of a sequence of interest, eg, chromosome 21, to a normalized sequence, eg, the chromosome 9 sequence tag density determined in a test sample. Similarly, “qualifying sequence dose” is a parameter that relates the sequence tag density of a sequence of interest to the sequence tag density of a normalized sequence determined in a qualified sample.

  As used herein, the term “sequence tag density” refers to the number of sequence reads mapped to a reference genome sequence, eg, the sequence tag density of chromosome 21 is mapped to chromosome 21 of the reference genome. The number of sequence reads generated by the sequencing method. As used herein, the term “sequence tag density ratio” means the ratio of the number of sequence tags mapped to a chromosome of a reference genome sequence, eg, chromosome 21, to the length of chromosome 21 in the reference genome.

  As used herein, the term “Next Generation Sequencing” refers to a sequencing method that is clonally amplified and capable of massively parallel sequencing on a single nucleic acid molecule. Examples are sequencing-by-synthesis using reversible dye terminators and sequencing-by-ligation.

  As used herein, the term “parameter” means a numerical value that characterizes a quantitative data set and / or a numerical relationship between quantitative data sets. For example, a ratio (or a function of the ratio) between the number of sequence tags mapped to a chromosome and the length of the chromosome to which the sequence tag is mapped is used as a parameter.

  As used herein, the terms “threshold” and “eligibility threshold” are arbitrary values that are calculated using the qualifying dataset and that serve as diagnostic limits for copy number variation in organisms, eg, aneuploidy Means the numerical value of If the result obtained from practicing the present invention exceeds a threshold, the subject can be diagnosed as having a copy number variation, eg, trisomy 21. Appropriate thresholds for the methods described in the present invention can be identified by analyzing normalized values (eg, chromosomal doses, NCVs, or NSVs) calculated for a training set of samples. The threshold can be identified using qualifying (ie, no anomaly) samples in a training set having both qualifying (ie, no anomaly) samples and anomalous samples. Use samples that are known to have chromosomal aneuploidy in the training set (ie, samples with anomalies) to see if the selected threshold is useful to distinguish the anomalies from the samples without anomalies in the test set (See the examples herein). The threshold selection depends on the confidence level of the user who wishes to perform classification. In some embodiments, the training set used to identify appropriate thresholds is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80. At least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, It shall have at least 3000, at least 4000, or more samples. Using a larger set of eligible samples is advantageous to improve the diagnostic utility of the threshold.

  As used herein, the term “normalized value” refers to the number of sequence tags identified for a sequence of interest (eg, a chromosome or chromosome fragment) and the normalized sequence (eg, normalized chromosome or normalized chromosome fragment). Means a numerical value associated with the number of sequence tags identified for. For example, a “normalized value” can be a chromosomal dose described elsewhere in this specification, or an NCV (Normalized Chromosome Value) described elsewhere in this specification, or elsewhere in this specification. The described NSV (Normalized Segment Value) can be used.

  As used herein, the term “read” refers to a DNA sequence of sufficient length (eg, at least about 30 bp) that can be used to identify larger sequences or regions, Aligned and specially assigned to chromosomes or genomic regions or genes.

  The term “sequence tag” herein is used interchangeably with “mapped sequence tag” and is specifically assigned by a larger sequence, eg, alignment to a reference genome, ie, mapped sequence reads. Means. The mapped sequence tag is uniquely mapped to the reference genome, ie assigned as a single location relative to the reference genome. Tags that can be mapped at more than one location relative to the reference genome, ie, tags that are not uniquely mapped, are not included in this analysis.

  As used herein, the terms “aligned”, “alignment”, or “aligning” are one or more identified as a match in the order of nucleic acid molecules to a known sequence from a reference genome. It means more array state. Such alignment can be done manually or, for example, Efficient Local Alignment of Nucleotide Data (ELAND) of nucleotide data delivered as part of a pipeline in Illumina Genomics Analysis. ) It can be done by computer algorithms including computer algorithms.

  As used herein, the term “reference genome” refers to any particular known genomic sequence (either partial or whole) in any organism or virus that can be used to refer to a sequence identified from a specimen. means. For example, reference genomes used for human specimens as well as many other organisms can be found at the National Center for Biotechnology Information (www.ncbi.nlm.hih.gov.). “Genome” means complete genetic information represented by a nucleic acid sequence in an organism or virus.

  As used herein, the term “clinically-relevant sequence” refers to a nucleic acid sequence known or suspected to be associated with or involved in a genetic disease or condition. Determining the presence or absence of clinically relevant sequences is useful in determining diagnosis or confirming diagnosis of a medical disease, or in making progress diagnosis of a disease.

  As used herein, the term “derived” as used in the context of a nucleic acid or nucleic acid mixture means that the nucleic acid was obtained from the source from which it originated. For example, in one embodiment, a nucleic acid mixture derived from two different genomes means that the nucleic acid, eg, cfDNA, is naturally released from the cell by a naturally occurring process such as necrosis or apoptosis. . In other embodiments, a mixture of nucleic acids derived from two different genomes means that the nucleic acids were extracted from two different types of cells from the specimen.

  The term “mixed sample” herein refers to a sample containing a mixture of nucleic acids from different genomes.

  As used herein, the term “maternal sample” refers to a biological sample taken from a pregnant specimen, eg, a female human.

  The term “biological fluid” as used herein means a fluid collected from a biological fluid source, such as blood, serum, plasma, sputum, exudate, cerebrospinal fluid, urine, semen, sweat, There are tears and saliva. As used herein, the terms “blood”, “plasma” and “serum” also include fractions or processed portions thereof. Similarly, when a sample is taken from a biopsy, cotton swab, smear, etc., the “sample” also includes a treated fraction or portion derived from a biopsy, cotton swab, smear, etc.

  As used herein, the terms “maternal nucleic acid” and “fetal nucleic acid” mean the nucleic acid of a pregnant female specimen and the nucleic acid of a fetus that the pregnant female specimen is in respectively.

  As used herein, the term “corresponds to” exists in different specimen genomes and does not necessarily have the same sequence in all genomes, but for example genetic information of a sequence of interest that is a gene or a chromosome. It means a nucleic acid sequence that is subject to other uniqueness, eg, a gene or a chromosome.

  As used herein, the term “substantially cell-free” encompasses the preparation of a desired sample that removes normally associated components from the desired sample. For example, a plasma sample is made substantially cell-free by removing blood cells associated with plasma, such as red cells (red blood cells). In some embodiments, the nearly cell-free sample is treated to remove cells that would otherwise contribute to the desired genetic material to be tested for CNV if not removed.

  As used herein, the term “fetal fraction” refers to the fraction of fetal nucleic acid present in a sample containing fetal and maternal nucleic acids.

  As used herein, the term “chromosome” refers to a gene carrier of a living cell that is derived from chromatin and contains DNA and protein components (histones). In this specification, the conventional internationally recognized individual human genome chromosome numbering system is adopted.

  The term “polynucleotide length” as used herein refers to the absolute number of nucleic acid molecules (nucleotides) in a sequence or region of a reference genome. The term “chromosome length” means the known length of a chromosome in base pairs, eg found on the World Wide Web at “genome.ucsc.edu/cgi-bin/hgTracks?Hgsid=167155613&chromInfoPage=” It is defined in the NCBI36 / hg18 assembly of the human chromosome.

  The term “specimen” as used herein also means human specimens as well as non-human specimens such as mammals, invertebrates, vertebrates, fungi, yeasts, bacteria, and viruses. Although the examples herein relate to humans and the terminology is primarily human-related, the concepts of the present invention can be applied to genomes from any plant or animal, and veterinary, animal science, laboratory, etc. It is useful in the field of

  As used herein, the term “disease state” refers to a “medical disease” in a broad sense, including all diseases and disorders, but affects an individual's health and benefits from medical assistance or medical treatment. Includes “injuries” that may be implemented and normal health conditions such as pregnancy.

  The term “complete” is used herein for chromosomal aneuploidy, which refers to a total chromosome excess or deficiency.

  The term “portion” used for chromosomal aneuploidy refers to an excess or deficiency in a portion of a chromosome.

  The term “mosaic” as used herein means that there are two cell populations with different karyotypes in one individual grown from a single fertilized egg. Mosaic phenomenon results from a growing mutation that only affects a small population of mature cells.

  As used herein, the term “non-mosaic” refers to an organism composed of cells of one karyotype, such as a human fetus.

  The term “using a chromosome” as used in determining chromosomal dose means herein using the sequence information obtained for a chromosome, ie the number of sequence tags obtained for the chromosome.

  As used herein, the term “sensitivity” is equal to the value obtained by dividing true positives by the sum of true positives and false negatives.

  As used herein, the term “specificity” is equal to the number of true negatives divided by the sum of true negatives and false positives.

  As used herein, the term “patient sample” means a biological sample obtained from a patient, ie, an individual who receives medical attention, care or treatment. The patient sample can be any sample described herein. Preferably, the patient sample is collected by a non-invasive procedure, for example a peripheral blood sample or a stool sample.

  As used herein, the term “hypodiploidy” means a chromosome number that is one or more than the normal half of the chromosome characteristics of the species.

DESCRIPTION The present invention has a mixture of nucleic acids from two different genomes and copies of different sequences of interest in a test sample known or suspected of differing total amounts in one or more sequences of interest. A method of determining copy number variation (CNV) is provided. Copy number polymorphisms determined by the method of the present invention include excess or deficiency of total chromosomes, alterations involving very large chromosomal fragments visible microscopically, DNA fragments ranging from kilobases (kb) to megabases (Mb) There is a large amount of supermicroscopic copy number polymorphism. The method of the present invention includes a statistical approach that is responsible for accrual variation stemming processing from interchromosomal and inter-sequence variation associated with the process. This method is applicable to determine CNVs in any fetal aneuploidy, and CNVs known or concerned about various medical disorders. CNV that can be determined by the method of the present invention includes trisomy and monosomy in any one or more of chromosomes 1-22 and X and Y, other chromosomal polysomy, and fragments in one or more of the chromosomes (Segment) deletions and / or duplications are included, which can be determined by sequencing the nucleic acid of the test sample only once. Any aneuploidy can be determined from the sequence information obtained by sequencing the nucleic acid of the test sample only once.

  CNV in the human genome has a major impact on human and diversity and disease predisposition (see Redon et al., Nature 23: 444-454 [2006], Shaikh et al. Genome Res 19: 1682-1690 [2009]) . CNV is implicated in genetic illnesses by different mechanisms and is often found to be due to an imbalance due to gene dosage or gene disruption. In addition to being directly related to genetic diseases, CNV has been able to mediate phenotypic abnormalities that can lead to disease. In recent years, several studies have reported a rare or de novo (new) CNV increase in complex diseases such as autism, ADHD and schizophrenia, compared to normal controls. It highlights the potential pathogenicity of a unique or unique CNV (see Sebat et al., 316: 445-449 [2007]; Walsh et al., Science 320: 539-543 [2008]). CNV arises from genomic rearrangements mainly due to deletions, duplications, insertions and unbalanced translocation events.

  The methods described herein employ next generation sequencing technology (NGS) to sequence clonally amplified DNA templates or single DNA molecules in parallel in a flow cell (eg, Volkerding et al., Clin Chem 55: 641-658 [2009]; Metzker M Nature Rev 11: 31-46 [2010]). In addition to high throughput sequence information, NGS provides quantitative information in that each sequence read is a countable “sequence tag” that represents an individual clone DNA template or a single DNA molecule. NGS sequencing techniques include pyrosequencing, sequencing-by-synthesis using reversible dye terminators, sequencing by oligonucleotide probe ligation, and ion semiconductor sequencing. is there. Sequencing DNA from individual samples individually (ie, single sequencing), or pooling DNA from multiple samples and sequencing as indexed genomic molecules in a single sequencing run (ie, run) Multiple sequencing) and can generate several hundred reads of the DNA sequence. Examples of sequencing techniques that can be used to obtain sequence information according to the method of the present invention are described below.

Sequencing Methods Some sequencing techniques are commercially available, for example, sequencing by hybridization platforms from Affymetrix Inc. (Sunnyvale, Calif.), And 454 Life Sciences (Life Sciences). [Sequence by synthesis base from [Bradford, Conn.]), Illumina / Solexa (Hayward, Calif.) And Helicos Biosciences (Cambridge, Mass.), And There is a sequencing by ligation platform from Applied Biosystems (Foster City, CA), which is described below. In addition to single molecule sequencing performed using Sequencing by Synthesis from Helicos Biosciences, other single molecule sequencing techniques include SMRT (registered trademark) from Pacific Biosciences. ), Ion Trent (R) technology, and for example, Nanopore (pore) sequencing developed by Oxford Nanopore Technologies. Although the automated Sanger method is seen as a “first generation” technology, Sanger sequencing, including automated Sanger sequencing, can also be used in the method of the present invention. Other sequencing methods include nucleic acid imaging (imaging) techniques, such as atomic force microscopy (AFM) or transmission electron microscopy (TEM). A typical sequencing technique is described below.

In one embodiment, the method of the present invention is tested using Helicos true single molecule sequencing (tSMS) technology (see Harris TD et al., Science 320: 106-109 [2008]). Obtain nucleic acid sequence information in the sample, eg, cfDNA in the maternal sample. In tSMS technology, a DNA sample is cleaved into strands of about 100-200 nucleotides and a poly A sequence is added to the 3 'end of each DNA strand. Each strand is labeled by the addition of a fluorescently labeled adenosine nucleotide. The DNA strands are then mated in the flow cell, which contains millions of oligo T capture sites that are immobilized on the flow cell surface. The template can have a density of about 1 billion templates / cm ² . The flow cell is attached to an instrument such as a HeliScope® sequencer, and a laser is irradiated on the surface of the flow cell to reveal the position of each template. The CCD camera can map the template position on the flow cell surface. Cleave and wash out the fluorescence level of the template. The sequencing reaction is initiated by introducing DNA polymerase and fluorescently labeled nucleotides. Acts as an oligo T nucleic acid primer. The polymerase incorporates labeled nucleotides into the primer according to template instructions. Remove the polymerase and unincorporated nucleotides. Templates that guide the incorporation of fluorescently labeled nucleotides are determined by imaging the flow cell surface. After imaging, the cleavage step removes the fluorescent label and the process is repeated for other fluorescently labeled nucleotides until the desired lead length is obtained. Sequence information is collected at each nucleotide addition step. Whole genome sequencing by single molecule sequencing technology eliminates PCR base amplification in sequencing library preparation and allows direct measurement of the sample itself rather than a copy measurement of the sample due to the directity of the sample preparation.

  In other embodiments, the method of the invention uses (Roche) 454 sequencing (see, eg, Margulies, M. et.al. Nature 437: 376-380 [2005]) to test samples. The sequence information of the nucleic acid, for example, cfDNA in the maternal test sample is obtained. 454 sequencing has two steps. In the first step, the DNA is cut into approximately 300-800 base pair fragments, which are rough at the ends. The oligonucleotide adapter is then ligated to the end of the fragment. The adapter acts as a primer for fragment amplification and sequencing. The fragment is attached to a DNA capture bead, such as a streptavidin-coated bead, for example using adapter B containing a 5 'biotin tag. Fragments attached to the beads are PCR amplified in oil-water emulsion droplets. This results in multiple copies of the clonally amplified DNA fragment in each bead. In the second step, the beads are captured in wells (picoliter size). Pyrosequencing is performed on each fragment in parallel. The addition of one or more nucleotides generates an optical signal that is recorded by the CCD camera of the sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated. Pyrosequencing utilizes pyrophosphate (PPi) that is released upon nucleotide addition. PPi is converted to ATP by ATP sulfurylation in the presence of adenosine 5 'phosphosulfate. Luciferase (a luminescent enzyme) uses ATP to convert luciferin to oxyluciferin, and this reaction generates light, which is measured and analyzed.

  In other embodiments, the methods of the present invention use SOLiD® technology (Applied Biosystems) to obtain nucleic acid sequence information in a test sample, eg, cfDNA in a maternal test sample. In SOLiD sequencing by ligation, genomic DNA is cut into fragments and adapters are attached to the 5 'and 3' ends of the fragments to generate a fragment library. Alternatively, adapters are attached to the 5 ′ and 3 ′ ends of the fragments, the fragments are arranged in a circle, the circularly arranged fragments are shortened to produce an internal adapter, and the resulting fragments 5 ′ and An internal adapter can be introduced by attaching an adapter to the 3 'end to create a matched pair library. A clonal bead population is then prepared in a microreactor containing beads, primers, templates, and PCR. Following PCR, the template is denatured and the beads are separated with an expanded template with increased beads. A 3 'modification is added to the template in the selected bead, which allows binding to the glass slide. The sequence consists of random oligonucleotides, in turn, partially hybridized and ligated with a central determinant base (or base pair) identified by a particular fluorophore (fluorescent dye molecule). Can be determined. After recording the color, the ligated oligonucleotide is cleaved and removed, and the process is then repeated.

  In other embodiments, the method of the present invention uses Pacific Biosciences' single molecule real time (SMRT) sequencing technology to obtain nucleic acid sequence information in a test sample, eg, cfDNA in a maternal test sample. In SMRT sequencing, continuous incorporation of dye-labeled nucleotides is imaged during DNA synthesis. A single DNA polymerase molecule is located on the bottom of an individual zero-mode wavelength detector (ZMW detector) that acquires sequence information while incorporated into the growing primer strand of nucleotides bound to phosphorus. Attach. ZMW is a confinement structure and can be observed against a background of fluorescent nucleotides that diffuse the incorporation of single nucleotides by DNA polymerase into and out of ZMW intensely (in milliseconds). It takes a few milliseconds for the nucleotide to be incorporated into the growing strand. During this time, the fluorescent label is excited to generate a fluorescent signal and the fluorescent tag is cleaved. The corresponding fluorescence measurement of the dye indicates which base has been incorporated. Repeat this process.

  In other embodiments, the method of the invention uses nanopore (pore) sequencing (see, eg, Soni GV and Meller A. Clin Chem 53: 1996-2001 [2007]) to sequence nucleic acids in a test sample. Obtain information, eg, cfDNA in a maternal test sample. Nanopore sequencing DNA analysis technology has been industrially developed by many companies, including Oxford Nanopore Technologies (Oxford, UK). Nanopore sequencing is a single molecule sequencing technique that allows direct sequencing when a single molecule of DNA is passed through the nanopore. Nanopores are pores on the order of 1 nanometer in diameter. By immersing the nanopore in a conductive fluid and applying a potential difference (voltage), ions are conducted to the nanopore to generate a slight current. The amount of current flowing is affected by the size and shape of the nanopore. As the DNA molecule passes through the nanopore, the nucleotides in the DNA molecule plug the nanopore to different extents and change the magnitude of the current flowing through the nanopore to different extents. Thus, the change in current as the DNA molecule passes through the nanopore represents the reading of the DNA sequence.

  In other embodiments, the methods of the present invention use chemically sensitive field effect transistor (chemFET) arrays (see, eg, US Patent Application Publication No. 20090026082) to determine the sequence information of nucleic acids in a test sample, such as a maternal test sample. To obtain the cfDNA. In one example of this technique, a DNA molecule is placed in a reaction chamber and a template molecule is mated to a sequencing primer bound to a polymerase. One or more triphosphates can be incorporated into a new nucleic acid strand at the 3 'end of the sequencing primer by a current change by chemFET. An array can have multiple chemFET sensors. In other embodiments, single nucleic acids can be attached to the beads, nucleic acids can be amplified on the beads, individual beads can be transferred to reaction chambers in the chemFET array (each chamber has a chemFET sensor), Nucleic acids can be sequenced.

  In another embodiment, the method of the invention uses nucleic acid sequence information in a test sample, eg, cfDNA in a maternal test sample, using Halcyon Molecular technology using a transmission electron microscope (TEM). Get. This method, known as Individual Molecule Placement Nano Transfer (IMPRNT), is a single-atom-resolution transmission electron that images high molecular weight (150 kb or more) DNA selectively labeled with heavy atom markers. Using a microscope, these molecules are arranged on an ultrathin film in a parallel array of ultra-dense (3 nm interstrand distance) so that the spacing between bases is constant. An electron microscope is used to image molecules on the film, determine the position of heavy atom markers, and extract base sequence information from DNA. This method is described in WO2009 / 046445. This method can sequence the complete human genome in less than 10 minutes.

  In another embodiment, the DNA sequencing technology is Ion Torrent single molecule sequencing, which combines semiconductor technology with simple sequencing chemistry to provide chemical encoding information (A, C, G , T) is directly translated into digital information (0, 1) on the semiconductor chip. In fact, hydrogen ions are released as a byproduct when nucleotides are incorporated into DNA strands by polymerases. Ion Trent uses a high-density array of microfabricated wells to perform this biochemical process in large quantities in parallel. Each well holds a different DNA molecule. An ion sensing layer is disposed below the well, and an ion sensor is disposed below the ion sensing layer. When a nucleotide, such as C, is added to the DNA template and then incorporated into the DNA strand, hydrogen ions are released. The charge from the ions changes the pH of the solution, and this change can be detected by an ion torrent ion sensor. This sequencer (basically the world's smallest solid state pH meter) determines the base and moves directly from chemical information to digital information. The ION Trent personal genome machine (PGM®) sequencer sequentially floods the chip with one nucleotide. If the next nucleotide that overflows the chip does not match, the voltage change is not recorded and the base is not determined. If there are two individual bases in the DNA strand, the voltage is doubled and the chip records the determined two individual bases. Nucleotide incorporation can be recorded in seconds by direct detection.

  In other embodiments, the methods of the invention use sequencing-by hybridization to obtain nucleic acid sequence information in a test sample, eg, cfDNA in a maternal test sample. Sequencing by hybridization involves contacting a plurality of polynucleotide sequences with a plurality of polynucleotide probes, each of which can optionally be anchored to a substrate. The substrate can be a flat surface having an array of known nucleotide sequences. Hybridization patterns to the array can be used to determine the polynucleotide sequence present in the sample. In other embodiments, each probe is anchored to a bead, such as a magnetic bead. Hybridization to the bead can be determined and used to identify multiple polynucleotide sequences within the sample.

  In other embodiments, the methods of the present invention use Illumina sequencing by synthesis and reversible terminator base sequencing chemistry (see, eg, Bentley et al., Nature 6: 53-59 [2009]). Mass sequence information of 1 million DNA fragments provides nucleic acid sequence information in the test sample, eg, cfDNA in the maternal sample. The template DNA can be genomic DNA, eg cfDNA. In some embodiments, cfDNA is used as a template and does not require fragmentation because cfDNA exists as a short fragment. For example, fetal cfDNA circulates in the bloodstream as a fragment of approximately 170 base pairs (bp) in length (see, eg, Fan et al., Clin Chem 56: 1279-1286 [2010]) prior to sequencing. DNA fragmentation is not required. Illumina sequencing technology relies on attaching fragmented genomic DNA to a planar, translucent surface that binds oligonucleotide anchors. The template DNA is end repaired to give a 5 'phosphorylated blunt end, and the polymerase activity of the Klenow fragment is used to add a single A base to the 3' end of the blunt phosphorylated DNA fragment. This addition prepares a DNA fragment for ligation to the oligonucleotide adapter, which has a single T base overhang at the 3 'end, increasing ligation efficiency. The adapter oligonucleotide is complementary to the flow cell anchor. Under limiting dilution conditions, adapter-modified, single-stranded template DNA is added to the flow cell and immobilized by hybridization to the anchor. The attached DNA fragments are expanded and bridge amplified to produce ultra-high density sequencing flow cells with hundreds of millions of clusters, each cluster containing 1000 copies of the same template. In one embodiment, randomly fragmented genomic DNA, such as cfDNA, is amplified using PCR before cluster amplification. Alternatively, non-amplified genomic library preparations are used to enrich for randomly fragmented genomic DNA, such as cfDNA, using only cluster amplification (eg, Kozarewa et al., Nature Methods 6: 291-295 [2009 ]reference). Templates are sequenced using a robust four-color DNA sequencing by synthesis technique using a reversible terminator with a removable fluorescent dye. High sensitivity fluorescence detection is achieved by using laser excitation and total internal reflection optics. Short sequence reads of about 20-40 bp align to a repetitively masked reference genome, and a unique mapping of short sequence reads to the reference genome is identified using specially developed data analysis pipeline software . A non-repetitive masked reference genome can also be used. Using either repetitive or non-repetitive masking reference genomes, only those reads that uniquely map to the reference genome are counted. After the first lead is completed, the template is regenerated in situ, allowing a second lead from the opposite end of the fragment. In this way, sequencing of either single end or repair end of the DNA fragment can be used. Partial sequencing of DNA fragments present in the sample is performed, and a sequence tag having a predetermined length of, for example, 36 bp is mapped to a known reference genome and counted. In one embodiment, the reference genomic sequence is the NCBI36 / hg18 sequence, and this sequence information is available from “genome.ucsc.edu/cgi-bin/hgTracks?Hgsid=167155613&chromInfoPage=” on the World Wide Web. As an alternative, the reference genome sequence is the GRCh37 / hg19 sequence, and this sequence information is available from “genome.ucsc.edu/cgi-bin/hgGateway” on the World Wide Web. Other public sequence information sources include GenBank, dbEST, dbSTS, EMBL (the European Molecular Biology Laboratory), and DDBJ (the DNA Databank of Japan). Many computer algorithms for aligning sequences are available, including but not limited to BLAST (altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), FASTA (Person & Lipman, 1988), BOWTIE (Langmead et al., Genome Biology 10: R25.1-R25.10 [2009]), or ELAND (Illumina, Inc., San Diego, CA, USA). In one embodiment, one end of a clonal expanded copy of plasma cfDNA is sequenced and processed by bioinformatics analysis for the Illumina Genome Analyzer, which includes nucleotides Use Efficient Large-Scale Alignment of Nucleotide Database (ELAND) software. In some embodiments of the methods described herein, the mapped sequence tag is about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp. About 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about It has a sequence read of 400 bp, about 450 bp, about 500 bp. Technological advances are expected that allow single end leads greater than 500 bp and allow for more than about 1000 bp when generating repair end leads. In one embodiment, the mapped sequence tag has a 36 bp sequence read. Sequence tag mapping is obtained by comparing the sequence of the tag to a reference sequence, thereby determining the chromosomal origin of the sequenced nucleic acid (eg, cfDNA) molecule, and no special genetic sequence information is required. . A slight degree of mismatch (0-2 mismatches per sequence tag) can also contribute to a small number of polymorphisms that may exist between the reference genome and the genome in the mixed sample.

Multiple sequence tags are obtained per sample. In some embodiments, at least about 3 × 10 ⁶ base sequence tags, at least about 5 × 10 ⁶ base sequence tags, having at least about 40 × 40 base pair (bp) reads, at least about 8 × 10 ⁶ base sequence tags, at least about 10 × 10 ⁶ base sequence tags, at least about 15 × 10 ⁶ base sequence tags, at least about 20 × 10 ⁶ base sequence tags, at least about 30 × 10 ^Six base sequence tags, at least about 40 × 10 ⁶ base sequence tags, at least about 50 × 10 ⁶ base sequence tags are obtained from mapping reads per sample to the reference genome. In one embodiment, all sequence reads are mapped to the entire region of the reference genome. In one embodiment, tags mapped to all regions in the reference genome, eg, all chromosomes, are counted, and CNVs in mixed DNA samples, ie sequences of interest, eg, excess or deficiency in a chromosome or part of a chromosome Determine the expression. This method does not require discrimination between the two genomes.

  The accuracy required for an accurate determination regarding whether CNV, eg aneuploidy is present or absent in a sample, is the variation in the number of sequence tags that map to the reference genome per sample (interchromosomal variation) in a single sequencing operation (run) , And variations in the number of sequence tags (inter-sequencing variation) that map to the reference genome in different sequencing operations (runs). For example, polymorphisms (variations) can be specifically declared for tags that map to GC rich or GC poor reference sequences. Other polymorphisms (variations) can arise from nucleic acid extraction and purification, sequencing library preparation, and using different procedures for different sequencing platforms. The method of the present invention uses sequence doses (chromosome doses, or fragment doses) based on knowledge about normalized sequences (normalized chromosomal sequences, or normalized fragment sequences) and is inherently interchromosomal (intra-run) and It is assumed to be a factor of the foreseeable variability of the stemming process between sequencing (between runs) and the base-dependent variability. Chromosome doses are based on knowledge of a normalized chromosome sequence that can be composed of a single chromosome or two or more chromosomes selected from chromosomes 1-22 and X and Y chromosomes. As an alternative, the normalized chromosomal sequence can consist of a single chromosomal fragment, or two or more fragments on one chromosome or on two or more chromosomes. The fragment dose can be composed of a single fragment of any one chromosome, or two or more fragments in any two or more chromosomes among chromosomes 1-22, X and Y. Based on knowledge of normalized fragment sequences.

Determination of normalized sequences (normalized chromosomal sequences and normalized fragments) in qualifying samples Normalized sequences are composed of cells of interest, eg cells with normal copy number for any one sequence in a chromosome or chromosomal fragment Identification using sequence information from a set of eligible samples taken from known specimens. The determination of the normalization sequence is described in steps 100, 120, 130, 140 and 145 in the embodiment of the method of the present invention shown in FIG. Sequence information taken from eligible samples is also used to determine a statistically significant identification of chromosomal aneuploidy in the test sample (see step 155 and examples in FIG. 1). FIG. 1 is a flowchart of an embodiment 100 according to the method of the invention for determining a CNV in a sequence of interest, eg, a chromosome or chromosome fragment, in a biological sample. In some embodiments, the biological sample comprises a mixture of nucleic acids taken from an analyte and involved by different genomes. Different genomes are involved in the sample by two individuals, for example, different genomes are involved by the fetus and the mother that held the fetus. In addition, the genome is involved in the sample by aneuploid cancer cells and euploid cells from the same specimen, for example from plasma samples of cancer patients.

  A set of qualified samples is taken to identify qualified normalization sequences and to obtain a variation value used to determine a statistically significant identification of CNV in the test sample. In step 110, a plurality of biologically qualified samples are taken from a plurality of analytes known to contain cells having normal copy numbers for any one sequence of interest. In one embodiment, the eligible sample is taken from a maternal body bearing a fetus that has been confirmed to have a normal chromosome copy number using cytogenetic means. The biologically qualified sample can be a biological fluid, such as plasma, or any suitable sample as described below. In some embodiments, a qualifying sample can include a mixture of nucleic acid molecules, eg, cfDNA molecules. In some embodiments, the eligible sample is a maternal plasma sample containing a mixture of fetal and maternal cfDNA molecules. Sequence information for normalized chromosomes and / or fragments of normalized chromosomes is obtained using any known sequencing method, for example, by sequencing at least a portion of fetal and maternal nucleic acids. Preferably, any one of the Next Generation Sequencing (NGS) methods described elsewhere herein are used to sequence fetal and maternal nucleic acids as single molecules or clonal amplified molecules. .

In step 120, at least a portion of each of all qualifying nucleic acids contained in the qualifying sample is sequenced, resulting in millions of sequence reads, eg, 36 bp reads that align, eg, with the reference genome of hg18. In some embodiments, the sequence reads are about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp. Technological advances are expected that allow single end leads greater than 500 bp, and even greater than about 1000 bp when generating repair end leads. In one embodiment, the mapped sequence tag has a 36 bp sequence read. Sequence reads align with the reference genome, and reads that are uniquely mapped to the reference genome are known as base sequence tags (sometimes abbreviated as “sequence tags”). In one embodiment, at least about 3 × 10 ⁶ qualified sequence tags, at least about 5 × 10 ⁶ qualified sequence tags, at least about 8 ×, having 20-40 base pair (bp) reads. 10 ⁶ qualified sequence tags, at least about 10 × 10 ⁶ qualified sequence tags, at least about 15 × 10 ⁶ qualified sequence tags, at least about 20 × 10 ⁶ qualified sequence tags, at least about 30 × 10 ⁶ One eligible sequence tag, at least about 40 × 10 ⁶ eligible sequence tags, at least about 50 × 10 ⁶ eligible sequence tags are obtained from reads that uniquely map to a reference genome.

  At step 130, all tags resulting from sequencing nucleic acids in the qualified sample are counted to determine the qualified sequence tag density. In one embodiment, the sequence tag density is determined as the number of eligible sequence tags that map to the sequence of interest in the reference genome. In other embodiments, the qualified sequence tag is determined as the number of qualified sequence tags mapped to the sequence of interest, normalized to the length of the targeted sequence of interest to be mapped. The sequence tag density determined as the ratio of tag density to sequence length of interest is referred to herein as the tag density ratio. Normalization to the sequence length of interest is not necessary and can be included as a step to reduce the number of digits in the numerical value for simplification for human interpretation. When all qualifying sequence tags are mapped and counted in each qualifying sample, the sequence tag density of the sequence of interest in the qualifying sample, eg, the clinically relevant sequence, is the sequence tag for additional sequences that later identify the normalized sequence Determined as density.

  In some embodiments, the sequence of interest is a chromosome associated with complete chromosomal aneuploidy, such as chromosome 21, and the qualified normalized sequence is not associated with chromosomal aneuploidy and is a polymorphism in sequence tag density ( Variation) is the complete chromosome that most closely approximates the sequence of interest sequence (ie, chromosome), eg, polymorphism of chromosome 21. Any one or more of chromosomes 1-22 and X and Y may be sequences of interest, and one or more chromosomes may be chromosomes 1-22, X and It can be identified as a normalized sequence for any one of the Y chromosomes. Normalized chromosomes can be individual chromosomes or can be a group of chromosomes as described elsewhere herein.

  In other embodiments, the sequence of interest is a partial aneuploidy, eg, a chromosomal fragment associated with a chromosomal deletion or insertion, or an unbalanced chromosomal translocation, and the normalized sequence is associated with a partial aneuploidy. In addition, the polymorphism (variation) in sequence tag density is the chromosome fragment that most closely resembles the polymorphism of the chromosome fragment associated with partial aneuploidy. Any one or more fragments in any one or more of chromosomes 1-22 and X and Y can be used as the sequence of interest.

  In all embodiments, a qualified normalized sequence is defined in a qualified sample depending on whether a single sequence or sequence group in the qualified sample is identified as a normalized sequence for any one or more sequences of interest. There is a variation in the sequence tag density that most closely approximates the determined sequence tag density of the sequence of interest. For example, a qualified normalized sequence is the sequence with the least variability (polymorphism), ie, the variability of the normalized sequence most closely approximates the variability of the sequence of interest.

  In some embodiments, the normalization sequence is the sequence that most distinguishes one or more eligible samples from one or more anomalous samples, and the normalization sequence is the most discriminative sequence. Meaning, i.e. the discriminability of the normalized sequence makes it possible to optimally discriminate against the sequence of interest in the anomaly test sample and easily distinguish the anomaly test sample from the other anomaly samples Can do. In other embodiments, the normalization sequence is the sequence with the least variability and the greatest discriminability. The level of discriminability is the statistics between sequence doses in the qualifying sample population, such as chromosomal or fragment doses, and chromosomal doses in one or more test samples, as described in the examples below. It can be determined as a scientific difference. For example, the discriminability can be expressed numerically as a T-test value, which is between the chromosomal dose in the population of eligible samples and the chromosomal dose in one or more test samples. Represents statistical differences. Alternatively, the discriminability can be expressed numerically as a Normalized Chromosome Value (NCV), which is a z-score for chromosomal doses as long as the NCV distribution is standard. Similarly, discriminability can be expressed numerically as a T-test value, which is the statistical difference between a fragment dose in a population of eligible samples and a fragment dose in one or more test samples. Represents. As an alternative, the discriminability of a fragment dose can be expressed numerically as a Normalized Segment Value (NSV), which is a z-score for a chromosomal dose as long as the NSV distribution is standard. In determining the z-score, the mean and standard deviation of chromosomal or fragment doses in the set of eligible samples can be used. Alternatively, the mean and standard deviation of chromosomal or fragment doses in a training set that includes eligible and anomalous samples can be used. In other embodiments, the normalization sequence is an sequence with minimal variability and maximum discriminability.

  The method of the present invention identifies sequences that have inherently similar characteristics and are susceptible to similar polymorphisms (variations) between samples and between sequencing tasks (runs), which can reduce sequence doses in test samples. Useful for making decisions.

Determining Sequence Dose (i.e. Chromosome Dose or Fragment Dose) in a Qualified Sample In step 140, based on the calculated qualifying tag density, a qualified sequence dose for the sequence of interest, i. Determined as the ratio of the sequence tag density to the qualifying sequence tag density of the additional sequence that identifies the normalized sequence in a later step 145 This is followed by using the identified normalized sequence to determine the sequence dose in the test sample.

  In one embodiment, the sequence dose in the eligible sample is a chromosome dose calculated as the ratio of the number of sequence tags for the chromosome of interest to the number of sequence tags for the normalized chromosomal sequence in the eligible sample. The normalized chromosomal sequence can be a single chromosome, a chromosomal group, a fragment of a single chromosome, or a group of fragments from different chromosomes. Therefore, the chromosomal dose related to the chromosome of interest is the ratio of the number of tags related to the chromosome of interest and the number of tags related to the normalized chromosome sequence composed of a single chromosome in the eligible sample, and (ii) the tag related to the chromosome of interest. The ratio of the number of tags to a normalized chromosome sequence composed of 2 or more chromosomes, (iii) the number of tags related to the chromosome of interest and the number of tags related to a normalized fragment sequence composed of a single chromosome fragment (Iv) the ratio of the number of tags for the chromosome of interest to the number of tags for a normalized fragment sequence composed of two or more fragments from one chromosome, or (v) the chromosome of interest A tag relating to a normalized fragment sequence composed of two or more chromosome fragments in two or more chromosomes As a ratio to the number. An example of determining the chromosomal dose for the chromosome 21 of interest according to (i) to (v) is shown below. Chromosome dose for the chromosome of interest, eg chromosome 21, the sequence tag density for chromosome 21 and the sequence tag density for each of the remaining chromosomes, ie, chromosomes 1-20, 22, 22 and X. (I) the chromosome dose for the chromosome of interest, eg chromosome 21, the sequence tag density for chromosome 21 and the sequence tag for all 2 or more possible combinations in the remaining chromosomes (Ii) determine the chromosome dose for the chromosome of interest, for example chromosome 21, as the ratio to the density, the ratio of the sequence tag density for chromosome 21 to the sequence tag density for fragments of other chromosomes, for example chromosome 9 (Iii) determine the chromosomal dose for the chromosome of interest, eg chromosome 21, Determined as the ratio of the row tag density to the sequence tag density for two fragments of another chromosome, for example two fragments of chromosome 9, (iv), and for the chromosome of interest, for example chromosome 21 Chromosome dose is determined as the ratio of the sequence tag density of chromosome 21 to the sequence tag density for two fragments of two different chromosomes, for example, the fragment of chromosome 9 and the fragment of chromosome 14.

  In other embodiments, the sequence dose in the qualified sample is the fragment dose calculated as the ratio of the number of sequence tags for the fragment of interest to the number of sequence tags for the normalized fragment sequence in the qualified sample. The normalized fragment sequence can be a single chromosomal fragment or a group of fragments from different chromosomes. Therefore, the fragment dose for the fragment of interest is the ratio of the number of tags for the fragment of interest to the number of tags for the normalized fragment sequence composed of a single fragment of the chromosome in the eligible sample, and (ii) for the fragment of interest. The ratio of the number of tags to the number of tags for a normalized fragment sequence composed of two or more fragments on one chromosome, or (iii) the number of tags for a fragment of interest is two or more different It is determined as the ratio to the tag number for a normalized fragment sequence composed of two or more fragments in the chromosome.

  Chromosome doses for one or more chromosomes of interest are determined in all eligible samples, and normalized chromosomal sequences are identified in step 145. Similarly, fragment doses for one or more fragments of interest are determined in all eligible samples, and normalized fragment sequences are identified in step 145.

Identifying normalized sequences from eligible sequence doses In step 145, the normalized sequence is identified as a sequence based on the sequence dose calculated for the sequence of interest, i.e. the variability in the sequence dose with respect to the sequence of interest across all eligible samples. Is minimized. The method of the present invention identifies sequences that have inherently similar characteristics and are susceptible to similar polymorphisms (variations) between samples and between sequencing tasks (runs), which can reduce sequence doses in test samples. Useful to decide

  Normalized sequences for one or more sequences of interest can be identified in a set of eligible samples, and then one or more in each test sample using the sequences identified in the eligible samples. The sequence dose for the sequence of interest is calculated (step 150) and the presence or absence of aneuploidy in each test sample is determined. Normalized sequences identified for the chromosome or fragment of interest may be used when using different sequencing platforms and / or when there is a difference in the purification of the nucleic acids to be sequenced and / or in the preparation of sequencing libraries. When there is a difference, it can be different. The use of normalized sequences according to the method of the present invention provides a special and sensitive criterion for copy number variation in chromosomes or fragments regardless of the sample preparation and / or sequencing platform used.

  In some embodiments, more than one normalized sequence can be identified, i.e., different normalized sequences can be determined for one sequence of interest, and multiple sequence doses can be determined for one sequence of interest. Can be decided on. For example, polymorphisms in the chromosomal dose for chromosome 21 of interest, eg, coefficient of variation, are minimal when using the sequence tag density of chromosome 14. However, two, three, four, five, six, seven, eight or more normalized sequences can be identified using the sequence dose determination for the sequence of interest in the test sample. . For example, the second dose for chromosome 21 in any one test sample can be determined using chromosome 7, 9, 11, or 12 as the normalized chromosome sequence, because This is because all these chromosomes have CVs similar to those of chromosome 14 (see Example 2, Table 2). Preferably, when a single chromosome is selected as the normalized chromosomal sequence for the chromosome of interest, the normalized chromosomal sequence is the chromosomal dose for the chromosome of interest that has the least variability across all samples being tested, eg, eligible samples. It becomes the chromosome which becomes.

Normalized Chromosomal Sequence as a Chromosomal Normalized Sequence In other embodiments, the normalized chromosomal sequence can be a single sequence or a sequence group. For example, in some embodiments, the normalized sequence is a sequence group, eg, a chromosomal group, which is identified as a normalized sequence for any one or more of chromosomes 1-22, X and Y. . Normalized sequence for the chromosome of interest, ie, the group of chromosomes containing the normalized chromosomal sequence is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 chromosomes, and the X and Y chromosomes One or both of them may or may not be included. The group of chromosomes identified as a normalized chromosomal sequence is the chromosome group that becomes the chromosomal dose for the chromosome of interest with minimal variability across all samples being tested, eg, eligible samples. Preferably, individual chromosomes and chromosome groups are tested together for the ability to best approximate the behavior of the sequence of interest selected as the normalized chromosomal sequence.

  In one embodiment, the normalized sequence of chromosome 21 is chromosome 9, chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, Select from chromosome 10, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, and chromosome 17. As an alternative, the normalized sequence of chromosome 21 is changed to chromosome 9, chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 10 A chromosome group selected from chromosome, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, and chromosome 17. In another embodiment, the chromosome group is a group selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14.

  In some embodiments, the method further comprises normalization determined by systematically calculating all chromosomal doses using each chromosome individually and all possible combinations with the remaining chromosomes. This is improved by using a sequence (see Example 7). For example, the normalized chromosomes systematically determined are, for each chromosome of interest, any one of chromosomes 1-22 and X and Y, and chromosomes 1-22, X and Y. It can be determined by systematically calculating all possible chromosomal doses using two or more combinations to determine which single chromosome or which chromosomal group is a normalized chromosome, Minimizes chromosomal dose variability with respect to the chromosome of interest across the set of eligible samples (see Example 7). Therefore, in one embodiment, the normalized chromosome sequence calculated systematically for chromosome 21 is a chromosome group consisting of chromosome 4, chromosome 14, chromosome 16, chromosome 20, and chromosome 22. A single chromosome or group of chromosomes can be determined for all chromosomes in the genome.

  In one embodiment, the normalized sequence of chromosome 18 is chromosome 8, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 9, chromosome 10, Select from chromosome 11, chromosome 12, chromosome 13, and chromosome 14. Preferably, the normalization sequence for chromosome 18 is selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14. As an alternative, the normalized sequence of chromosome 18 is changed from chromosome 8, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 9, chromosome 10, chromosome 11, A chromosome group selected from chromosome, chromosome 12, chromosome 13, and chromosome 14. Preferably, the chromosome group is a group selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.

  In other embodiments, the normalized sequence of chromosome 18 uses each possible normalized chromosome individually (as described elsewhere herein) and all possible combinations of normalized chromosomes. It is determined by systematically calculating all possible chromosomal doses. Therefore, in one embodiment, the normalized sequence for chromosome 18 is a normalized chromosome composed of a chromosome group consisting of chromosome 2, chromosome 3, chromosome 5, and chromosome 7.

  In one embodiment, the normalized sequence of chromosome X is chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, 10 Chromosome, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15 and chromosome 16 are selected. Preferably, the X chromosome normalization sequence is selected from chromosome 2, chromosome 3, chromosome 5, chromosome 6 and chromosome 8. As an alternative, the normalized sequence of chromosome X is the chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11. Chromosome group selected from chromosome 12, chromosome 13, chromosome 14, chromosome 15, and chromosome 16. Preferably, the chromosome group is a group selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

  In other embodiments, the normalization sequence of the X chromosome uses each of the possible normalization chromosomes and possible normalization chromosome combinations of normalization chromosomes (as described elsewhere herein). Thus, all possible chromosomal doses are determined by systematic calculation. Therefore, in one embodiment, the normalized sequence of the X chromosome is a normalized chromosome consisting of a group of chromosomes 4 and 8.

  In one embodiment, the normalized sequence of chromosome 13 is chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, Select from chromosome 11, chromosome 12, chromosome 14, chromosome 18, and chromosome 21. Preferably, the normalized sequence for chromosome 13 is a chromosome selected from chromosome 2, chromosome 3, chromosome 5, chromosome 6, and chromosome 8. In another embodiment, the normalized sequence of chromosome 13 is the chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10. Chromosome group selected from the 11th chromosome, the 12th chromosome, the 14th chromosome, the 18th chromosome, and the 21st chromosome. Preferably, the chromosome group is a group selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

  In other embodiments, the normalization sequence of chromosome 13 uses each of the possible normalization chromosomes and the possible normalization chromosome combinations of normalization chromosomes (as described elsewhere herein). Thus, all possible chromosomal doses are determined by systematic calculation. Accordingly, in one embodiment, the normalized sequence of chromosome 13 is a normalized chromosome having a group of chromosomes 4 and 5. In another embodiment, the normalized sequence of chromosome 13 is a normalized chromosome consisting of a group of chromosomes 4 and 5.

  There are more than 30 polymorphisms in the chromosomal dose of the Y chromosome, and each of these normalized chromosomes is used independently to determine the Y chromosome dose. Therefore, any one chromosome or a group of two or more chromosomes selected from chromosomes 1-22 and X is used as the normalization sequence for the Y chromosome. In one embodiment, the at least one normalized chromosome is a chromosome group consisting of chromosomes 1 to 22 and the X chromosome. In another embodiment, the chromosome group is a chromosome group consisting of chromosome 2, chromosome 3, chromosome 4, chromosome 5, and chromosome 6.

  In other embodiments, the normalized sequence of the Y chromosome uses each of the possible normalized chromosomes and possible normalized chromosome combinations of the normalized chromosomes (as described elsewhere herein). Thus, all possible chromosomal doses are determined by systematic calculation. Therefore, in one embodiment, the normalized sequence of the Y chromosome is a normalized chromosome having a chromosome group consisting of chromosomes 4 and 6. In another embodiment, the normalized sequence of the Y chromosome is a normalized chromosome composed of a chromosome group consisting of chromosomes 4 and 6.

  The normalization sequence used to calculate the dose on different chromosomes of interest or different fragments of interest should be the same normalization sequence or different for different chromosomes of interest or different fragments of interest It can be a normalized array. For example, the normalized sequence (single or group) of chromosome C of interest can be the same or different from the normalized sequence (single or group) of chromosome C of interest, for example.

  The normal sequence of a complete chromosome can be a complete chromosome or a complete chromosome group, or it can be a chromosome fragment or a group of one or more chromosome fragments.

Normalized Fragment Sequence as a Chromosomal Normalized Sequence In other embodiments, the chromosomal normalized sequence can be a normalized fragment sequence. The normalized fragment sequence can be a single fragment or a group of fragments of one chromosome or can be fragments from two or more different chromosomes. Normalized fragment sequences can be determined by systematically calculating all combinations of fragment sequences in the genome. For example, the normalized fragment sequence of chromosome 21 can be a single fragment larger or smaller than the size of chromosome 2, chromosome 2 is about 47 Mbp (million base pairs), and about 140 Mbp of chromosome 9 and Are different in size. As an alternative, the normalized sequence for chromosome 21 can be a combination of the sequence from chromosome 1 and the sequence from chromosome 12.

  In one embodiment, the normalized sequence of chromosome 21 is a fragment of chromosomes 1 to 20, chromosome 22, X and Y, or a normalized fragment sequence of two or more fragment groups. In another embodiment, the normalization sequence of chromosome 18 is a fragment or group of chromosomes 1 to 17, chromosomes 19 to 22, X and Y. In another embodiment, the normalized sequence of chromosome 13 is a fragment or group of chromosomes 1-12, 14-22, X and Y. In another embodiment, the normalized sequence of the X chromosome is a fragment or group of chromosomes 1 to 22 and the Y chromosome. In another embodiment, the normalized sequence of the Y chromosome is a fragment or group of chromosomes 1 to 22 and the X chromosome. A normalized fragment sequence of a single fragment or group of fragments can be determined for every chromosome in the genome. Two or more fragments of the normalized fragment sequence can be fragments from one chromosome, or two or more fragments can be fragments of two or more chromosomes. As explained for the normalized chromosomal sequence, the normalized fragment sequence can be the same for two or more different chromosomes.

Normalized fragment sequence as a normalized sequence for a chromosomal fragment The presence or absence of CNV of a sequence of interest can be determined when the sequence of interest is a chromosomal fragment. Chromosomal fragment copy number polymorphism can determine the presence or absence of partial chromosomal aneuploidy. Described below are examples of partial chromosomal aneuploidy associated with various fetal abnormalities and medical conditions. Chromosome fragments can be of any length. For example, the length can range from kilobases to hundreds of megabases. The human genome exists over 3 billion DNA bases, and the DNA bases are divided into tens, thousands, hundreds of thousands, and millions of fragments of different sizes, and these fragments of different sizes are processed by the method of the present invention Can be determined by. The normalized sequence of the chromosomal fragment is a single fragment from any one of chromosomes 1-22 and X and Y, or any one or more of chromosomes 1-22, X and Y. A normalized fragment sequence that can be a fragment group.

  A normalized sequence of a fragment of interest is a sequence that has variability across the chromosome and variability across samples that are close to the sequence of the fragment of interest. Normalized sequence determination relates to determining the normalized sequence of the chromosome of interest when the normalized sequence is any one or more fragment groups of chromosomes 1-22, X and Y. It can be executed as described. A normalized fragment sequence of a fragment or fragment group uses all possible combinations in one fragment and two or more fragments to determine that the eligible sample, ie the fragment of interest, is diploid. Can be identified by calculating the fragment dose as the normalized sequence of the fragment of interest in each sample in the set of known samples, and the normalized sequence is all qualified as described above for the normalized chromosomal sequence. Determine that the fragment variability of interest across the sample provides the lowest fragment dose.

  For example, for a fragment of interest of 1 Mb (megabase), the remaining 3 million fragments (minus 1 mg of the fragment of interest) in the approximately 3 Gb human genome can be used individually or in combination with each other in a set of eligible samples The fragment dose of the fragment of interest can be calculated to determine which single fragment or group of fragments will serve as the normalized fragment sequence for eligible and test samples. Fragments of interest can vary from about 1000 bases to as much as tens of megabases. A normalized fragment sequence can be composed of one or more fragments of the same size as the sequence of interest. In other embodiments, the normalized fragment sequence may be composed of fragments that are different from and / or different from the fragments of the sequence of interest. For example, a normalized fragment sequence of a sequence having a length of 10,000 bases can be 20,000 bases in length, and has a sequence combination of different lengths, eg, 7,000 + 8,000 + 5,000 bases. be able to. As described elsewhere herein for normalized chromosomal sequences, normalized fragment sequences are used to identify each possible normalized chromosomal fragment in the normalized fragment individually and in all possible combinations (normalized). Can be determined by systematically calculating all possible chromosomal and / or fragment doses (as described elsewhere herein for chromosomal sequences). A single fragment or fragment group can be determined for every fragment and / or chromosome in the genome.

  The normalization sequences used to calculate the dose in different chromosomal fragments of interest can be the same or can be different normalization sequences for different chromosomal fragments of interest. For example, the normalization sequence of the normalization fragment (single or group) of the chromosome fragment A of interest is the same, or the normalization sequence is different from the normalization fragment (single or group) of the chromosome fragment B of interest, for example. be able to.

Aneuploidy determination in a test sample Based on the identification of normalized sequences in a qualified sample, sequence against a sequence of interest in a test sample having a nucleic acid mixture derived from a genome that differs in one or more sequences of interest Determine the dose.

  In step 115, a test sample is taken from a specimen suspected or known to have clinically relevant CNV in the sequence of interest. The test sample can be a biological fluid, such as plasma, or any suitable sample as described below. In some embodiments, the test sample will comprise a mixture of nucleic acid molecules, eg, cfDNA molecules. In some embodiments, the test sample is a maternal plasma sample containing a mixture of fetal and maternal cfDNA molecules.

At step 125, at least a portion of the test nucleic acid in the test sample is sequenced as described for the qualified sample to generate millions of sequence reads, eg, 36 bp reads. Similar to step 120, the reads resulting from sequencing the nucleic acids in the test sample are uniquely mapped to a reference genome. As described for step 120, at least about 3 × 10 ⁶ qualified sequence tags, at least about 5 × 10 ⁶ qualified sequence tags, having at least 20-40 base pair (bp) reads, at least About 8 × 10 ⁶ qualified sequence tags, at least about 10 × 10 ⁶ qualified sequence tags, at least about 15 × 10 ⁶ qualified sequence tags, at least about 20 × 10 ⁶ qualified sequence tags, at least about 30 X10 ⁶ qualified sequence tags, at least about 40 x 10 ⁶ qualified sequence tags, at least about 50 x 10 ⁶ qualified sequence tags are obtained from reads that uniquely map to a reference genome.

  At step 135, all tags resulting from sequencing nucleic acids in the test sample are counted to determine the test sequence tag density. In one embodiment, the number of test sequence tags mapped to the sequence of interest is normalized to the known length of the sequence of interest mapped to obtain a test sequence tag density ratio. As described for the qualified sample, it is not necessary to normalize to the known length of the sequence of interest and can be included as a step to reduce the number of digits in the number for simplification for human interpretation. When counting all mapped test sequence tags in a test sample, the sequence tag density of a sequence of interest in the test sample, eg, a clinically relevant sequence, is added corresponding to at least one normalized sequence identified in a qualified sample Determined as the sequence tag density for the target sequence.

  At step 150, a test sequence dose is determined for the sequence of interest in the test sample based on the identification of at least one normalized sequence in the eligible sample. As described throughout the specification, at least one normalized sequence can be a single sequence or a sequence group. The sequence dose of the sequence of interest in the test sample is the ratio of the sequence tag density determined for the sequence of interest in the test sample to the sequence tag density of at least one normalized sequence determined in the test sample, where The normalized sequence in the test sample corresponds to the normalized sequence identified in the eligible sample for the particular sequence of interest. For example, if the normalized sequence identified for chromosome 21 in a qualified sample is determined to be a chromosome, eg, chromosome 14, the test sequence dose for chromosome 21 (the sequence of interest) is the sequence of chromosome 21 It is determined as a ratio between the tag density and the sequence tag density of chromosome 14 determined in each test sample. Similarly, chromosome doses of chromosome 13, chromosome 18, X chromosome, Y chromosome and other chromosomes related to chromosome aneuploidy are determined. The normalized sequence of the chromosome of interest can be a single chromosome or group of chromosomes, or a single chromosome fragment or group of chromosome fragments. As mentioned above, the sequence of interest can be part of a chromosome, for example part of a chromosome fragment. Thus, the chromosomal fragment dose can be determined as the ratio of the sequence tag density determined for the fragment in the test sample to the normalized chromosomal fragment sequence tag density in the test sample, in which case the normalized in the test sample Fragments correspond to normalized fragments (single fragments or fragment groups) identified in a qualified sample for a particular fragment of interest. Chromosome fragments range in size from kilobases to megabases.

  In step 155, a threshold is derived from the qualifying sequence dose determined for the plurality of qualifying samples and the standard deviation established for the sequence dose determined for the sample with known aneuploidy with respect to the sequence of interest. The exact classification depends on the difference between the probability distributions for different classes, i.e. aneuploidy types. Preferably, the threshold is selected from an empirical distribution of each type of aneuploidy, eg trisomy 21. Trisomy 13 as described in the examples illustrating the use of the method of the invention to determine chromosomal aneuploidy by sequencing cfDNA extracted from a maternal sample containing a mixture of fetal and maternal nucleic acids. There are possible thresholds established to classify aneuploidy of trisomy 18, trisomy 21, trisomy 21 and X monosomy. The threshold value determined to distinguish anomalous samples of a certain chromosomal aneuploidy may be the same or different from the threshold value determined to distinguish anomalous samples of different aneuploidy. As shown in the examples, the threshold for each chromosome of interest is determined from the variability of the chromosomal dose across samples and between sequencing runs. The less variability of the chromosomal dose of any chromosome of interest is, the less the variance in the chromosomal dose of interest across all non-invariant samples used to set a threshold for determining different aneuploidies. Narrow.

  At step 160, the copy number variation of the sequence of interest is determined by comparing the test sequence dose of the sequence of interest with at least one threshold established from the qualified sequence dose.

  In step 165, the calculated dose of the test sequence of interest is compared to the dose set as the threshold selected according to the user-defined confidence threshold, and the sample is classified as “normal”, “abnormal”, “no call”. A “no call” sample is a sample that cannot make a definitive diagnosis reliably.

  Other embodiments of the invention provide methods for prenatal diagnosis of fetal chromosomal aneuploidy in biological samples having fetal and maternal nucleic acid molecules. This diagnosis involves obtaining sequence information that sequences at least a portion of a mixture of fetal and maternal nucleic acid molecules taken from a biological test sample, eg, a maternal plasma sample, and from the sequence data one or more chromosomes of interest. Computerized normalization doses and / or normalized fragment doses of one or more fragments of interest, and chromosomal doses of interest chromosomes and / or fragment doses of fragments of interest in the test sample Based on determining a statistically significant difference from the established threshold in the (normal) sample and making a prenatal diagnosis based on the statistically significant difference. As described in step 165 of the method of the present invention, a normal or abnormal diagnosis is performed. “No call” is given when a normal or abnormal diagnosis cannot be made with certainty.

Sample CNV, eg, a sample used to determine chromosomal aneuploidy and partial aneuploidy, is present in a cell or has “cell-free” nucleic acid. In some embodiments of the invention, it is advantageous to harvest cell-free nucleic acids, such as cell-free DNA (cfDNA). Cell-free nucleic acids, including cell-free DNA, can be collected from biological samples such as, but not limited to, plasma and serum by various methods known in the art (Chen et al., Nature Med. 2: 1033-1035 [ 1996]; Lo et al., Lancet 350: 485-487 [1997]). Fractionation, centrifugation (eg, density gradient centrifugation), DNA-specific precipitation, or high throughput cell sorting and / or separation methods can be used to separate cell-free DNA from cells.

  The sample with the nucleic acid mixture to which the method according to the invention is applied is a biological sample such as a tissue sample, a biological fluid sample or a cell sample. In some embodiments, the nucleic acid mixture is purified or separated from the biological sample by any one of the known methods. The sample can be composed of purified or separated polynucleotides, or can include biological samples such as tissue samples, biological fluid samples, or cell samples. Biological fluids include, but are not limited to, blood, plasma, serum, sweat, tears, sputum, urine, semen, inner ear fluid, lymph fluid, saliva, cerebrospinal fluid, exudate, bone marrow suspension, vaginal fluid, transcervical canal There are lavage fluid, brain fluid, ascites, breast milk, respiratory organs, intestinal and urogenital secretions, amniotic fluid, and leukocyte symbiosis samples. In some embodiments, the sample is a sample that can be easily collected by non-invasive procedures, such as blood, plasma, serum, sweat, tears, sputum, urine, semen, inner ear fluid, saliva, or feces. Preferably, the biological sample is a peripheral blood sample or a plasma and serum fraction. In other embodiments, the biological sample is a swab or smear sample, a biopsy sample, or a cell culture. In other embodiments, the sample is a mixture of two or more biological samples, eg, the biological sample comprises two or more of a biological fluid sample, a tissue sample, and a cell culture sample. can do. As used herein, the terms “blood”, “plasma” and “serum” also include fractions or processed portions thereof. Similarly, when a sample is taken from a biopsy, cotton swab, smear, etc., the “sample” also includes a treated fraction or portion derived from a biopsy, cotton swab, smear, etc.

  In some embodiments, the sample can be taken from a source, including but not limited to different individuals, different developmental stages of the same or different individuals, individuals with different diseases (eg, having cancer or having a genetic disease Individuals suspected to be), normal individuals, samples taken at different stages of the disease in individuals, samples from individuals who have received different environmental factors, or individuals with a pathological predisposition, or infectious disease agent (HIV) There are samples from individuals exposed to.

  In one embodiment, the sample is a pregnant female body, eg, a maternal sample taken from a pregnant woman. In this case, the sample is analyzed using the methods described herein for prenatal diagnosis of fetal potential chromosomal abnormalities. The maternal sample can be a tissue sample, a biological fluid sample, or a cell sample. Non-limiting examples of biological fluids include blood, plasma, serum, sweat, tears, sputum, urine, semen, inner ear fluid, lymph fluid, saliva, cerebrospinal fluid, rupture fluid, bone marrow suspension, vaginal fluid, There are transcervical lavage fluid, brain fluid, ascites, breast milk, respiratory organs, intestinal and urogenital secretions, amniotic fluid, and leukocyte symbiosis samples. In other embodiments, the maternal sample can be a mixture of two abnormal biological samples, eg, the biological sample is two or more of a biological fluid sample, a tissue sample, and a cell culture sample. It can have the above. In some embodiments, the sample is a sample that can be easily collected by non-invasive procedures, such as blood, plasma, serum, sweat, tears, sputum, urine, semen, inner ear fluid, saliva, or feces. Preferably, the biological sample is a peripheral blood sample or a plasma and serum fraction. In other embodiments, the biological sample is a swab or smear sample, a biopsy sample, or a cell culture. As used herein, the terms “blood”, “plasma” and “serum” also include fractions or processed portions thereof. Similarly, when a sample is taken from a biopsy, cotton swab, smear, etc., the “sample” also includes a treated fraction or portion derived from a biopsy, cotton swab, smear, etc.

  Samples can be taken from tissues, cells, or polynucleotides containing sources cultured in vitro. Cultured samples include, but are not limited to, cultures (eg, tissues or cells) maintained at different media and conditions (eg, pH, pressure, or temperature), cultures (eg, tissue maintained for different lengths of time) Or cells), cultures (eg, tissues or cells) treated with different factors or reagents (eg, drug candidates or modulators), or sources containing different types of cultures in tissues or cells.

  Methods for isolating nucleic acids from biological sources are known and will vary based on the nature of the source. One skilled in the art can readily isolate nucleic acids from the sources required for the methods described in the methods. In some embodiments, it is advantageous to fragment nucleic acid molecules in a nucleic acid sample. When performing fragmentation, it can be random or special, for example using a restriction endonuclease. Random fragmentation methods are known in the art and include, for example, deoxyribonuclease digestion, alkaline treatment, and physical shearing. In one embodiment, the sample nucleic acid is taken from unfragmented cfDNA. In other embodiments, the sample nucleic acid is taken from genomic DNA fragmented into fragments of about 500 base pairs or more and to which the NGS method can be easily applied.

CNV Determination for Prenatal Diagnosis Cell-free fetal DNA and RNA circulating in maternal blood provide both maternal management and reproductive decision support, and therefore, early prenatal diagnosis (NIPD) for many genetic diseases diagnosis). The existence of cell-free DNA circulating in the bloodstream has been known for 50 years. More recently, the presence of small amounts of circulating fetal DNA has been found in maternal blood flow during pregnancy (see Lo et al., Lancet 350: 485-487 [1997]). Cell-free fetal DNA (cfDNA), believed to be derived from dying placental cells, has been found to be composed of short fragments, typically less than 200 bp in length ( Chan et al., Clin Chem 50: 88-92 [2004]) and this cfDNA can be confirmed early in the fourth week of pregnancy (Illanes et al., Early Human Dev 83: 563-566 [2007] ) And have been found to be removed from the maternal circulation within a few hours of distribution (see Lo et al., Am J Hum Genet 64: 218-224 [1999]). In addition to CfDNA, a cell-free fetal RNA (cfRNA) fragment can be identified in the maternal bloodstream, which is derived from a gene transcribed in the fetus or placenta. Extraction of these fetal genetic elements from maternal blood samples and subsequent analysis provides new NIPD opportunities.

  The method of the present invention is a polymorphism-independent method that can be used for NIPD and can determine fetal aneuploidy without the need to distinguish fetal cfDNA from maternal cfDNA. In some embodiments, the aneuploidy is full chromosomal trisomy or monosomy or partial trisomy or monosomy. Partial aneuploidy is caused by a deficiency or excess of part of the chromosome and includes chromosomal imbalances caused by unstable translocations, unstable inversions, deletions and insertions. To date, the most commonly known viable aneuploidy is trisomy 21 or Down's syndrome (DS), which is caused by the presence of some or all of chromosome 21. DS is caused by genetic or sporadic abnormalities, and an extra copy of all or part of chromosome 21 attaches to another chromosome (usually chromosome 14) to form a single deformed chromosome. DS is associated with excess mortality resulting from intellectual disability, serious learning difficulties, and long-term health problems such as heart disease. Other aneuploidies known to be clinically significant include Edwards syndrome (Trisomy 18) and Pateau syndrome (Trisomy 13), often dying at the first few months of life To do. Abnormalities related to the number of sex chromosomes are also known, including X monosomy, such as Turner syndrome (XO), and triple X syndrome (XXX), Kleinfelter syndrome (XXY), and XYY syndrome in boys Related to various phenotypes, including lack and decline of intellectual skills These and other chromosomal abnormalities can be diagnosed prenatally using the methods of the present invention.

  According to some embodiments of the present invention, the trisomy determined by the method of the present invention is Trisomy 21 (T21; Down's Syndrome), Trisomy 18 (T18; Edwards Syndrome), Trisomy 16 (T16), 22 Trisomy (T22; Cat Eye Syndrome), Trisomy 15 (T15; Praderwilli Syndrome), Trisomy 13 (T13; Patou Syndrome), Trisomy 8 (T8; Workany Syndrome), XXY (Kleinfelter Syndrome), XYY, or XXX Not limited to trisomy. According to the teachings of the present invention, various other complete and partial trisomy can be determined in fetal cfDNA. Examples of partial trisomy include, but are not limited to, partial trisomy lq32-44, 9p trisomy, 4th trisomy mosaic, 17p trisomy, partial trisomy 4q26-qter, 9th trisomy, partial 2p trisomy, partial trisomy 1q, and / or There is partial trisomy 6p / monosomy 6q.

  The method of the present invention can determine X-chromosome monosomy and partial monosomy. Examples of the partial monosomy include 13 monosomy, 15 monosomy, 16 monosomy, 21 monosomy, and 22 monosomy. Is known to be found in miscarriage pregnancy. A partial monosomy of a chromosome generally found in complete aneuploidy can also be determined by the method of the present invention. Monosomy 18p is a rare chromosomal disorder that lacks all or part of the short arm of chromosome 18 (monochromic). This disorder is typically characterized by dwarfism, varying degrees of intellectual developmental dysfunction, delayed speech development, skull and facial (craniofacial) region malformations, and / or additional physical abnormalities. The craniofacial disorders vary widely from case to case with respect to scope and severity. Symptoms caused by the structure of chromosome 15 or copy number polymorphism include Angelman syndrome and Praderwilli syndrome, which have a deficiency in gene activity in the same part of chromosome 15, 15q11 to q13. Some translocations and microdeletions may be asymptomatic for the carrier parent, but still develop large genetic disorders in the offspring. For example, a healthy mother with 15q11-q13 microdeletions can give birth to a child with Angelman syndrome, a severe neurodegenerative disease. The method of the present invention can be used to identify such partial and other deletions in the fetus. Partial monosomy 13q is a rare chromosomal disorder, and is a result of the loss of part of the long arm in chromosome 13 (monochromic). A fetus born with partial monosomy 13q exhibits low birth weight, head and face deformities (craniofacial region), skeletal abnormalities (particularly hands and feet), and other physical abnormalities. Mental retardation is a characteristic of this symptom. Mortality during childhood is higher for individuals born with this disorder. Almost all cases of partial monosomy 13q occur randomly (sporadic) for no obvious reason. The 22q11.2 deletion syndrome (also known as DiGeorge syndrome) is a syndrome that develops due to the deletion of a small piece of chromosome 22. The (22q11.2) deletion occurs near the middle of the chromosome in the long arm of one of the chromosome pairs. The characteristics of this syndrome vary widely among members of the same syndrome, and affect many parts of the body. Characteristic signs and symptoms are birth defects, such as congenital heart disease, most commonly related to defects in the palate associated with neuromuscular closure problems (palatopharyngeal insufficiency), learning disabilities, minor differences in facial features And recurrent infections. A microdeletion in chromosomal region 22q11.2 is associated with a risk of increasing schizophrenia by 20 to 30 fold. In one embodiment, the method of the present invention is used to determine a partial monosomy including, but not limited to, monosomy 18p, partial monosomy of chromosome 15 (15q11-q13), partial monosomy 13q, and part of chromosome 22 Monosomy can also be determined using the method of the present invention.

  The method of the invention can also be used to determine any aneuploidy when one parent is known to be such an abnormal carrier. These aneuploidies include, but are not limited to, a mosaic of small supermarker marker chromosomes (SMC), t (11; 14) (p15; p13) translocation, and unbalanced translocation t (8; 11) ( p23.2; p15.5), 11q23 microdeletion, 17p11.2 deletion, 22q13.3 deletion, Xp22.3 microdeletion, 10p14 deletion, 20p microdeletion, DiGeorge syndrome [del (22) (q11.2q11.23), Williams syndrome (7q11.23 and 7q36 deletion), 1p36 deletion, 2p microdeletion, neurofibromatosis type 1 (17q11.2 microdeletion), Yq deletion, Wolf Wolf-Hirschhorn syndrome (WHS, 4p16.3 microdeletion), 1p36.2 microdeletion, 11q14 deletion, 19q13.2 microdeletion, Rubinstein Taibi (16p13.3 microdeletion) 7p21 microdeletion Deletion, Miller-Dieker syndrome, 17p11.2 deletion, and 2q37 microdeletion .

Determination of Complete Fetal Chromosome Aneuploidy In one embodiment, the present invention determines the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acid molecules. Provide a method. Suitably, the method of the invention determines the presence or absence of any four or more complete chromosomal aneuploidies. The steps of the method of the present invention include (a) obtaining sequence information of fetal and maternal nucleic acids in a maternal test sample, and (b) using chromosome information from chromosomes 1-22 and X and Y chromosomes. Identifying the number of sequence tags in each of any one or more selected chromosomes of interest and identifying the number of sequence tags of the normalized chromosomal sequence in each of any one or more chromosomes of interest. Have. The normalized chromosomal sequence can be a single chromosome or a chromosome group selected from chromosomes 1-22 and X and Y chromosomes. The method of the invention further uses in step (c) the number of sequence tags identified for each of any one or more chromosomes of interest and the number of sequence tags identified for each normalized chromosomal sequence. A single chromosomal dose in each of any one or more chromosomes of interest, and each single chromosomal dose in each of any one or more of the chromosomes of interest in step (d) Compare to the threshold of each of the one or more chromosomes of interest, thereby determining the presence or absence of any one or more different complete fetal aneuploidies in the maternal test sample.

  In some embodiments, step (c) comprises the step of identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each chromosome of interest, and the sequence identified for the normalized chromosome of each chromosome of interest. Calculate as a ratio to the number of tags.

  In another embodiment, step (c) comprises the step of identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each chromosome of interest, and the sequence identified for the normalized chromosome for each chromosome of interest. Calculate as a ratio to the number of tags. In other embodiments, step (c) comprises calculating the sequence tag ratio of the chromosome of interest by associating the number of sequence tags obtained for the chromosome of interest with the length of the chromosome of interest, Corresponding normalized chromosomal sequence tag number is related to normalized chromosomal sequence length, and chromosomal dose of the chromosome of interest is calculated as the ratio of the sequence tag density of the chromosome of interest to the sequence tag density of the normalized sequence By doing. Repeat this calculation for every chromosome of interest. Steps (a)-(d) can be repeated for test samples from different maternal specimens.

  Examples of embodiments in which four or more complete fetal chromosomal aneuploidies are determined in a maternal test sample comprising a mixture of fetal and maternal cell-free DNA include: (a) fetal and maternal cell-free DNA in the test sample Sequencing at least a portion of the molecule to obtain sequence information; (b) using this sequence information, any 20 or more selected from chromosomes 1-22, X and Y chromosomes Identifying the number of sequence tags for each of the chromosomes of interest and identifying the number of sequence tags for the normalized chromosome of each of the 20 or more chromosomes of interest; (c) the 20 or more of the interests Using the number of sequence tags identified for each of the target chromosomes and the number of sequence tags identified for each normalized chromosome, the 20 or more Calculating a single chromosome dose for each of the target chromosomes; and (d) comparing each single chromosome dose for each of the 20 or more chromosomes of interest to a threshold of each of the 20 or more chromosomes of interest. And thereby determining the presence or absence of any 20 or more different complete fetal chromosomal aneuploidies in the test sample.

  In other embodiments, a method for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in the maternal test sample described above uses a normalized fragment sequence that determines the dose in the chromosome of interest. To do. In this case, the method comprises: (a) obtaining sequence information of fetal and maternal nucleic acids in the test sample; and (b) using this sequence information from chromosomes 1-22, X and Y. Identifying the number of sequence tags for each of any one or more selected chromosomes of interest and identifying the number of sequence tags for the normalized fragment sequence of each of the one or more selected chromosomes of interest. . A normalized fragment sequence can be a single fragment of a chromosome or a group of fragments from any one or more different chromosomes. The method further comprises (c) using the number of sequence tags identified for each of the one or more chromosomes of interest and the number of sequence tags identified for each normalized fragment sequence, Calculating a single chromosome dose for each of any one or more chromosomes of interest; and (d) any one single chromosome dose for each of the one or more chromosomes of interest Or comparing to a threshold value for each of the more or less chromosomes of interest and thereby determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in the test sample.

  In some embodiments, step (c) has identified a single chromosomal dose for each chromosome of interest relative to the number of sequence tags identified for each chromosome of interest and the normalized fragment sequence for each chromosome of interest. Calculated as a ratio to the number of sequence tags.

  In other embodiments, step (c) comprises calculating the sequence tag ratio of the chromosome of interest by associating the number of sequence tags obtained for the chromosome of interest with the length of the chromosome of interest, The number of tags in the corresponding normalized fragment sequence is related to the length of the normalized fragment sequence, and the chromosomal dose of the chromosome of interest is expressed as the ratio of the sequence tag density of the chromosome of interest to the sequence tag density of the normalized fragment sequence. Do by calculating. Repeat this calculation for every chromosome of interest. Steps (a)-(d) can be repeated for test samples from different maternal specimens.

Means for comparing chromosomal doses of different sample sets can be obtained by determining a normalized chromosome value (NCV) that relates the chromosomal doses in the test sample to the average of the corresponding chromosomal doses in the qualified sample set. it can. NCV is calculated as follows:
here,
Are the estimated mean and standard deviation for chromosome jose in the eligible sample set, respectively, and x _ij is the observed chromosome jose in test sample i.

  In some embodiments, the presence or absence of at least one complete fetal chromosomal aneuploidy is determined. In other embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, Determining whether or not at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 complete fetal chromosomal aneuploidy within a sample; In this case, the 22 complete fetal chromosomal aneuploidy corresponds to the complete chromosomal aneuploidy in any one or more autosomes, the 23rd chromosome aneuploidy and the 24th chromosome aneuploidy are Corresponds to the complete fetal chromosomal aneuploidy of the X and Y chromosomes. Since sex chromosome aneuploidy includes tetrasomy, pentasomy and other polysomy, the number of different complete chromosomal aneuploidies that can be determined by the method of the present invention is at least 24, at least 25, at least 26, at least 27, There are at least 28, at least 29, or at least 30 complete chromosomal aneuploidies. Thus, the number of different complete fetal chromosomal aneuploidies determined is related to the number of chromosomes of interest selected for analysis.

  In one embodiment, determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in the maternal test sample as described above was selected from chromosomes 1-22, X and Y. The normalized fragment sequence of one chromosome of interest is used. In other embodiments, two or more chromosomes of interest are assigned to No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10, Select from any two or more of chromosomes 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, chromosomes X and Y To do. In one embodiment, any one or more chromosomes of interest selected from chromosomes 1-22, X and Y are at least 20 chromosomes selected from chromosomes 1-22, X and Y. Determine the presence or absence of at least 20 different complete fetal chromosomal aneuploidies. In other embodiments, any one or more chromosomes of interest selected from chromosomes 1-22, X and Y are all of chromosomes 1-22, X and Y, and In this case, the presence or absence of complete fetal chromosome aneuploidy in all of chromosomes 1-22 and X and Y is determined. Different complete fetal chromosomal aneuploidies that can be determined include complete chromosomal trisomy, complete chromosomal monosomy, and complete chromosomal polysomy. Examples of complete fetal chromosomal aneuploidy include trisomy in any one or more of the autosomes, such as trisomy 2, trisomy 8, trisomy 9, trisomy 21, trisomy 13, trisomy 16, Without limiting to trisomy 18 and trisomy 22, sex chromosome trisomy, such as 47, XXY, 47XXX, and 47XYY; sex chromosome tetrasomy, such as 48, XXYY, 48, XXY, and 48XXXY, and sex Pentasomy, such as 49, XXXXYY, 49, XXXXY, and 49, XXXXXX, and 49, XYYYY; and X monosomy. Other complete fetal chromosomal aneuploidies that can be determined by the method of the present invention are described below.

Determination of Partial Fetal Chromosome Aneuploidy In another embodiment, the present invention determines the presence or absence of any one or more different partial fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acid molecules. Provide a way to do it. The steps of the method of the present invention are as follows: (a) obtaining sequence information of fetal and maternal nucleic acids in a maternal test sample; and (b) using the sequence information, chromosomes 1-22 and X and Y chromosomes. Identifying the number of sequence tags in each of any one or more fragments in any one or more chromosomes of interest selected from and any one in any one or more chromosomes of interest Or identifying the number of sequence tags of the normalized fragment sequence in each of the more fragments. Normalized fragment sequences can be single fragments of chromosomes or groups of fragments from one or more different chromosomes. The method of the present invention further comprises, in step (c), the number of sequence tags identified for each of any one or more fragments in any one or more chromosomes of interest, and each normalized fragment sequence Calculate the single fragment dose for each of any one or more fragments in any one or more chromosomes of interest using the number of sequence tags identified for and optionally in step (d) Each single fragment dose in any one or more fragments in each of the one or more chromosomes of interest of any one or more chromosome fragments in any one or more of the chromosomes of interest To determine the presence or absence of any one or more different partial fetal chromosomal aneuploidies in the maternal test sample.

  In some embodiments, step (c) comprises converting any one or more single fragment doses in any one or more chromosomes of interest into any one or more chromosomes of interest. For the number of sequence tags identified for each of any one or more fragments and the normalized fragment sequence of each of any one or more fragments in any one or more chromosomes of interest Calculated as the ratio to the number of sequence tags identified.

  In another embodiment, step (c) comprises calculating the sequence tag ratio of the fragment of interest by associating the number of sequence tags obtained for the fragment of interest with the length of the fragment of interest and The number of tags in the corresponding normalized fragment sequence is related to the length of the normalized fragment sequence, and the fragment dose of the fragment of interest as Do by calculating. Repeat this calculation for every chromosome of interest. Steps (a)-(d) can be repeated for test samples from different maternal specimens.

Means for comparing fragment doses of different sample sets can be obtained by determining a normalized segment value (NSV) that relates the fragment doses in the test sample to the average of the corresponding fragment doses in the eligible sample set. it can. NSV is calculated as follows:
here,
Are the estimated mean and standard deviation for the jth fragment dose in the eligible sample set, respectively, and x _ij is the observed jth fragment dose in test sample i.

  In some embodiments, the presence or absence of at least one partial fetal chromosomal aneuploidy is determined. In other embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more partial fetal chromosomal aneuploidies Presence / absence is determined within one sample. In one embodiment, one fragment of interest selected from any one of chromosomes 1-22, X, and Y is selected from chromosomes 1-22, X, and Y. In another embodiment, two or more fragments of interest selected from chromosomes 1-22, X chromosome and Y chromosome are numbered 1, 2, 3, 4, 5, 6, 7 No. 8, No. 9, No. 10, No. 11, No. 12, No. 13, No. 14, No. 15, No. 16, No. 17, No. 18, No. 19, No. 20, No. 21, No. 22 chromosome, X chromosome And any two or more of the Y chromosomes. In one embodiment, any one or more fragments of interest selected from Chromosomes 1-22, X and Y are at least one selected from Chromosomes 1-22, X and Y, Have five, ten, fifteen, twenty, twenty-five or more fragments, in which case at least one, five, ten, fifteen, twenty, twenty-five different partial fetal chromosomes Determine the presence or absence of aneuploidy. Different partial fetal chromosomal aneuploidies that can be determined include partial duplication, partial growth, partial insertion, and partial deletion. Examples of partial fetal chromosomal aneuploidies include autosomal partial monosomy and partial trisomy. As the autosomal partial monosomy, the partial monosomy of chromosome 1, the partial monosomy of chromosome 5, the partial monosomy of chromosome 7, the partial monosomy of chromosome 7, the partial monosomy of chromosome 11, the partial monosomy of chromosome 15, There is a partial monosomy of chromosome 17, a partial monosomy of chromosome 18, and a partial monosomy of chromosome 22. Other partial fetal chromosome aneuploidies that can be determined by the method of the present invention are described below.

  In any one of the embodiments described above, the test sample is a maternal sample selected from blood, plasma, serum, urine, and saliva samples. In some embodiments, the maternal sample is a plasma sample. The nucleic acid molecule of the maternal sample is a mixture of fetal and maternal cell-free DNA molecules. Nucleic acid sequencing can be performed using Next Generation Sequencing (NGS), as described elsewhere herein. In other embodiments, the sequencing is massively parallel sequencing using sequencing by synthesis with a reversible dye terminator. In other embodiments, the sequencing is sequencing by ligation. In still other embodiments, the sequencing is single molecule sequencing. Optionally, an amplification step is performed prior to sequencing.

CNV determination of clinical disorders In addition to the early determination of birth defects, the methods described herein can be applied to any abnormality determination in the representation of gene sequences in the genome.

  Plasma and serum DNA in blood from cancer patients contains measurable amounts of tumor DNA, which can be recovered and used as a surrogate source of tumor DNA, and tumors can contain gene sequences or whole Even in the chromosome, it is characterized by aneuploidy or inappropriate numbers. A determination of the difference in a given sequence in a sample from an individual, i.e. the total amount of the sequence of interest, can be used to diagnose a medical disease. In some embodiments, the methods of the invention can be used to determine the presence or absence of chromosomal aneuploidy in a patient suspected or known to suffer from cancer. Furthermore, the method of the invention determines the presence or absence of a disease state, determines the presence or absence of a pathogen, such as a viral nucleic acid, determines a chromosomal abnormality associated with graft versus host disease (GVHD), And can also be used to determine individual involvement in forensic analysis.

  Embodiments of the present invention comprise a mixture of nucleic acids from two different genomes and include a sequence of interest in a test sample known or suspected of having a difference in the total amount of one or more sequences of interest, eg, clinically relevant A method for evaluating copy number variation of a sequence is provided. The nucleic acid mixture is derived from two or more cell types. In one embodiment, the nucleic acid mixture is derived from normal and cancerous cells taken from a specimen suffering from a medical disease, such as cancer.

  The onset of cancer is often accompanied by variations in the number of whole chromosomes, ie, complete chromosomal aneuploidy, and / or changes in the number of chromosome fragments, ie, partial aneuploidy, which are chromosomal instability ( It is caused by a process known as CIN (chromosome instability) (see Thomas et al., Swiss Med Weekly 2011: 141: w13170). Many solid tumors, such as breast cancer, progress as metastases from the initial location by the accumulation of several genetic abnormalities (Sato et al., Cancer Res., 50: 7184-7189 [1990]; Jongsma et.al., J Clin Pathol: Mol Path 55: 305-309 [2002]). Such genetic abnormalities, when accumulated, result in proliferative predominance, genetic instability, and the accompanying ability to rapidly develop drug resistance, increased angiogenesis, proteolysis and metastasis. This genetic abnormality results in the effect of either a “tumor suppressor gene” that declines or a predominantly acting oncogene. Deletions and genetic recombination leading to loss of heterozygosity (LOH) are believed to play a major role in tumor progression by not covering mutant tumor suppressor alleles.

  cfDNA has been found in the blood circulation of patients diagnosed with malignant tumors, including but not limited to lung cancer (see Pathak et al., Clin Chem 52: 1833-1842 {2006]), prostate (See Schwartzenbach et al., Clin Cancer Res 15: 1032-8 [2009]) and breast cancer (see Schwartzenbach et al., Breast-cancer-research.com/content/11/5/R7 [2009]) Has been found. Identification of cancer-related genomic instabilities that can be determined with circulating cfDNA in cancer patients is a potential diagnostic and predictive tool. In one embodiment, the method of the invention is suspected or known to be afflicted with cancer, eg, epithelial malignant, non-epithelial malignant, lymphoma, leukemia, germ cell tumor, and blastoma. Evaluate the CNV of interest in a sample having a nucleic acid mixture derived from an analyte. In one embodiment, the sample is a plasma sample collected (processed) from peripheral blood, the plasma sample comprising a mixture of cfDNA derived from normal and cancerous cells. In other embodiments, the biological sample required to determine whether CNV is present is other biological fluid, such as, but not limited to, serum, sweat, tears, sputum, urine, semen, inner ear Fluid, lymph, saliva, cerebrospinal fluid, rupture fluid, bone marrow suspension, vaginal fluid, transcervical lavage fluid, brain fluid, ascites, breast milk, respiratory organs, intestinal and genitourinary tract secretions, amniotic fluid, and leukocyte symbiosis samples Or from a mixture of cancerous and non-cancerous cells from a tissue biopsy, swab or smear sample.

  A sequence of interest is a nucleic acid sequence that is known or suspected to play a role in the development and / or progression of cancer. Examples of sequences of interest are nucleic acid sequences, ie complete chromosomes and / or chromosomal fragments, which are amplified or deleted in cancerous cells as described below.

  In one embodiment, the methods of the invention can be used to determine the presence or absence of chromosomal amplification. In some embodiments, chromosomal amplification is the overproduction of one or more chromosomes. In other embodiments, chromosomal amplification is the overproduction of one or more chromosomal fragments. In still other embodiments, chromosomal amplification is the overproduction of two or more fragments on two or more chromosomes. Chromosomal amplification is the overproduction of one or more oncogenes.

  Genes that are predominantly associated with human solid tumors are effective as overexpression or phenotypic expression. Gene amplification is a common mechanism towards upregulation of gene expression. Evidence from studies on cell development indicates that over 50% of human breast cancers produce large amplifications. Most notably, the cell surface is amplified by the amplification of human epidermal growth factor receptor 2 (HER2) in humans against the proto-oncogene located on chromosome 17 (17 (17q21-q22)). Overexpression of HER2 in humans and generate excessive and unregulated signals in breast cancer and other malignancies (see Park et al., Clinical Breast Cancer 8: 392-401 [2008]). Various oncogenes have been found to be amplified in other human malignancies. Amplification of cellular oncogenes in human tumors includes c-myc, promyelocytic leukemia cell line HL60 and small cell lung cancer cell lines, primary neuroblastoma (stage III and IV), neuroblastoma cell lines , Retinoblastoma cell lines, primary tumors, small cell lung cancer lines, and N-myc in tumors, small cell lung cancer cell lines and L-myc in tumors, acute myeloid leukemia, and c- in colon cancer cell lines myb, c-erbb in epidermoid carcinoma cells and primary glioma, c-K-ras-2 in primary epithelial malignancies of lung, colon, bladder and rectum, N in breast epithelial malignant cell lines with amplification of -ras (Varmus H., Ann Rev Genetics 18: 553-612 (1984) [cited in Watson et al., Molecular Biology of the Gene (4th ed .; Benjamin / Cummings Publishing Co. 1987)] ).

  In one embodiment, the method of the invention can be used to determine the presence or absence of a chromosomal deletion. In some embodiments, the chromosomal deletion is a loss of one or more whole chromosomes. In other embodiments, the chromosomal deletion is a loss of one or more fragments on one chromosome. In still other embodiments, the chromosomal deletion is a loss of two or more fragments on two or more chromosomes. A chromosomal deletion can include the loss of one or more tumor suppressor genes.

  Chromosomal deletions, including tumor suppressor genes, play an important role in the development and progression of solid tumors. The retinoblastoma suppressor gene (Rb-1) located on chromosome 13q14 is the most strongly characterized tumor suppressor gene. The 105 kDa nuclear phosphoprotein, the Rb-1 gene product, plays an important role in cell cycle regulation (see Howe et al., Proc Natl Acad Sci (USA) 87: 5883-5887 [1990]). Rb protein alteration or loss expression results from inactivation of both alleles, either by point (abrupt) mutations or chromosomal deletions. Changes in the Rb-i gene are not only in retinoblastoma but also in other malignant tumors such as osteosarcoma, small cell lung cancer (see Rygaard et al., Cancer Res 50: 5312-5317 [1990]), and breast cancer. I know that there is. Restriction fragment length polymorphism (RFLP) studies have shown that these tumor types are often heterozygous at 13q and one of the Rb-1 alleles is a gross chromosomal deletion (See Bowcock et al., Am J Hum Genet, 46:12 [1990]). Chromosome 1 abnormalities, including duplications, deletions and imbalanced translocations involving Chromosome 6 and other partner chromosomes, include regions of Chromosome 1, particularly 1q21-1q32 and 1p11-13, which are myeloproliferative It shows having an oncogene or tumor suppressor gene that is etiologically related to both the chronic and advanced stages of the organism (see Caramazza et al., Eur J Hematol 84: 191-200 [2010]). Myeloproliferative neoplasms are also associated with deletion of chromosome 5. Complete loss or interstitial deletion of chromosome 5 is the most common karyotypic abnormality in myelodysplastic syndromes (MDS). Individual del (5q) / 5q MDS patients have a better prognosis than patients with additional karyotype defects, and patients with additional karyotype defects are myeloproliferative neoplasms (MPNs) and Tend to develop acute myeloid leukemia. The frequency of unbalanced chromosome 5 deletion is based on the idea that 5q has one or more tumor suppressor genes that have a fundamental role in the growth control of hematopoietic stem / progenitor cells (HSC / HPC). It reaches. Cytogenetic mapping of commonly deleted regions (CDRs) centered on 5q31 and 5q32 is a candidate tumor suppressor gene comprising ribosome subunit RPS14, transcription factor Egrl / Krox20, cytoskeletal reconstitution protein and α-catenin (See Eisenmann et al., Oncogene 28: 3429-3441 [2009]). Cytogenetic and allelotyping studies of fresh tumors and tumor cell lines have shown that allelic deletions from several distinct regions of chromosome 3 including 3p25, 3p21-22, 3p21.3, 3p12-13 and 3p14 (allele) The earliest and most frequent genome is found in severe, wide-ranging epithelial cancers in the lung, breast, kidney, head and neck, ovary, neck, colon, pancreas, esophagus, bladder, and other organs That's it. Several tumor suppressor genes are mapped to the 3p region of the chromosome, and it is believed that stroma deletion or promoter hypermethylation precedes the loss of 3p or entire chromosome 3 in the development of epithelial malignancies (See Angeloni D., Briefings Functional Genomics 6: 19-39 [2007]). Neonates and children with Down syndrome (DS) often present with congenital transient leukemia and an increased risk of acute myeloid leukemia and acute lymphoblastic leukemia. Chromosome 21 with about 300 genes includes many structural abnormalities, such as translocations, deletions, and amplifications, in leukemias, lymphomas, and solid tumors. Furthermore, the gene located on chromosome 21 was identified to play an important role in tumorigenesis. Chromosome 21 abnormalities related to somatic cells and structurally are associated with leukemia, and specific genes including RUNX1, TMPRSS2 and TFF located at 21q play a role in tumorigenesis (Fonatsch C Gene Chromosomes Cancer 49: 497-508 [ 2010]).

In one embodiment, the method of the present invention provides a means for assessing the association between gene amplification and tumor progression. The correlation between amplification and / or deletion and cancer stage or grade is important for prediction, because such information contributes to the definition of genetic tumor grade, which is the worst Better predict future disease progression in more advanced tumors with the prediction of In addition, information regarding initial amplification and / or deletion is useful in assessing these events as predictors of future disease progression. As identified by the method of the present invention, gene amplification and deletion can be achieved by other known parameters such as tumor grade, tissue structure, Brd / Urd labeling index, hormone status, lymph node metastasis, tumor size, Can be related to survival and other tumor characteristics obtained from epidemiological and biostatistical studies. For example, tumor DNA to be tested by the method of the present invention includes atypical hyperplasia, noninvasive ductal carcinoma, stage I-III cancer, and metastatic lymph nodes, and the relationship between amplification and deletion and stage Sex identification can be performed. The associations that have been made can provide the most effective therapeutic intervention possible. For example, a region that is constantly amplified contains an overexpressed gene, and the product of this gene can be therapeutically attacked (eg, growth factor receptor tyrosine kinase, p185 ^HER2 ).

  Identifying amplification and / or deletion events related to drug resistance by determining the copy number variation of the nucleic acid sequence of cancer cells that have metastasized from the primary cancer to other sites using the method of the present invention. Can do. If gene amplification and / or deletion is the development of karyotypic instability that allows rapid development of drug resistance, the tumor from patients who are resistant to chemotherapy rather than the tumor of patients who are sensitive to chemotherapy More amplification and / or deletion is expected in the primary tumor. For example, if amplification of a particular gene contributes to the development of drug resistance, the region surrounding that gene is constantly amplified with tumor cells from pleural effusion rather than the primary tumor of a patient resistant to chemotherapy It is expected that. Finding an association between gene amplification and / or deletion and drug resistance development allows patient identification of whether postoperative adjuvant therapy is beneficial.

  Similar to that described for determining the presence or absence of complete and / or partial fetal chromosomal aneuploidy in a maternal sample, any patient sample containing nucleic acid, eg, DNA or cfDNA, using the method of the present invention. The presence or absence of complete and / or partial chromosomal aneuploidy (including patient samples that are not maternal samples) can be determined. The patient sample can be any biological sample, as described elsewhere herein. Preferably, the sample is taken by a non-invasive procedure. For example, the sample can be a blood sample or blood serum and plasma fraction. Alternatively, the sample can be a urine sample or a stool sample. In yet other embodiments, the sample is a tissue biopsy sample. In all cases, the sample contains nucleic acid, such as cfDNA or genomic DNA, which is purified and sequenced using any NGS sequencing as described above.

  Both complete and partial chromosomal aneuploidy associated with formation and cancer progression can be determined by the method of the present invention.

Determining Complete Chromosome Aneuploidy in a Patient Sample In one embodiment, the present invention provides a method for determining the presence or absence of any different one or more complete chromosomal aneuploidies in a patient test sample comprising a nucleic acid molecule. To do. In some embodiments, the methods of the invention determine the presence or absence of any one or more different complete chromosomal aneuploidies. The steps of the method of the present invention include (a) obtaining sequence information of patient nucleic acid in a patient test sample, and (b) selecting from chromosomes 1 to 22, X chromosome and Y chromosome using the sequence information. Identifying the number of sequence tags in any one or more chromosomes of interest and identifying the number of sequence tags of normalized chromosomal sequences in any one or more chromosomes of interest. The normalized chromosomal sequence can be a single chromosome or a chromosome group selected from chromosomes 1-22 and X and Y chromosomes. The method of the invention further uses in step (c) the number of sequence tags identified for each of any one or more chromosomes of interest and the number of sequence tags identified for each normalized chromosomal sequence. A single chromosomal dose for each of any one or more chromosomes of interest and a single chromosomal dose for each of any one or more of the chromosomes of interest in step (d). Compare to the threshold of each of the one or more chromosomes of interest, thereby determining the presence or absence of any one or more different complete patient aneuploidies in the patient test sample.

  In some embodiments, step (c) comprises identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each chromosome of interest, and a sequence identified for the normalized chromosome for each chromosome of interest. Calculate as a ratio to the number of tags.

  In another embodiment, step (c) comprises the step of identifying a single chromosomal dose for each chromosome of interest, the number of sequence tags identified for each chromosome of interest, and the sequence identified for the normalized chromosome for each chromosome of interest. Calculate as a ratio to the number of tags. In other embodiments, step (c) comprises calculating the sequence tag ratio of the chromosome of interest by associating the number of sequence tags obtained for the chromosome of interest with the length of the chromosome of interest, Corresponding normalized chromosomal sequence tag number is related to normalized chromosomal sequence length, and chromosomal dose of the chromosome of interest is calculated as the ratio of the sequence tag density of the chromosome of interest to the sequence tag density of the normalized sequence By doing. Repeat this calculation for every chromosome of interest. Steps (a)-(d) can be repeated for test samples from different patients.

  Examples of embodiments for determining one or more complete chromosomal aneuploidies in a cancer patient test sample comprising a cell-free DNA molecule include: (a) at least a portion of a patient's cell-free DNA molecule in the test sample; Sequencing to obtain sequence information; (b) using this sequence information, each of any 20 or more selected chromosomes of interest selected from chromosomes 1-22, X and Y. Identifying a sequence tag number and identifying a normalized chromosome sequence tag number for each of the 20 or more chromosomes of interest; (c) for each of the 20 or more chromosomes of interest Using each identified sequence tag number and the identified sequence tag number for each normalized chromosome, each of the 20 or more chromosomes of interest alone Calculating a chromosomal dose; and (d) comparing each single chromosomal dose of each of the 20 or more chromosomes of interest to a threshold of each of the 20 or more chromosomes of interest, and thereby Determining the presence or absence of any 20 or more different complete chromosomal aneuploidies in the patient test sample.

  In other embodiments, the method for determining the presence or absence of any one or more different complete chromosomal aneuploidies in the patient test sample described above uses a normalized fragment sequence that determines the dose in the chromosome of interest. . In this case, the method comprises (a) obtaining the sequence information of the nucleic acid in the test sample, and (b) using the sequence information, any one selected from chromosomes 1 to 22, the X chromosome and the Y chromosome. Identifying the number of sequence tags for each of the one or more chromosomes of interest and identifying the number of sequence tags for the normalized fragment sequence of each of the one or more chromosomes of interest. A normalized fragment sequence can be a single fragment of a chromosome or a group of fragments from any one or more different chromosomes. The method further comprises (c) using the number of sequence tags identified for each of the one or more chromosomes of interest and the number of sequence tags identified for each normalized fragment sequence, Calculating a single chromosome dose for each of any one or more chromosomes of interest; and (d) any one single chromosome dose for each of the one or more chromosomes of interest Or comparing to a threshold for each of the more or less chromosomes of interest and thereby determining the presence or absence of any one or more different complete chromosomal aneuploidies in the patient test sample.

  In some embodiments, step (c) has identified a single chromosomal dose for each chromosome of interest relative to the number of sequence tags identified for each chromosome of interest and the normalized fragment sequence for each chromosome of interest. Calculated as a ratio to the number of sequence tags.

  In other embodiments, step (c) comprises calculating the sequence tag ratio of the chromosome of interest by associating the number of sequence tags obtained for the chromosome of interest with the length of the chromosome of interest, The number of tags in the corresponding normalized fragment sequence is related to the length of the normalized fragment sequence, and the chromosomal dose of the chromosome of interest is expressed as the ratio of the sequence tag density of the chromosome of interest to the sequence tag density of the normalized fragment sequence. Do by calculating. Repeat this calculation for every chromosome of interest. Steps (a)-(d) can be repeated for test samples from different patients.

Means for comparing chromosomal doses of different sample sets can be obtained by determining a normalized chromosome value (NCV) that relates the chromosomal doses in the test sample to the average of the corresponding chromosomal doses in the qualified sample set. it can. NCV is calculated as follows:
here
Are the estimated mean and standard deviation for chromosome jose in the eligible sample set, respectively, and x _ij is the observed chromosome jose in test sample i.

  In some embodiments, the presence or absence of at least one complete chromosomal aneuploidy is determined. In other embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, Determining the presence or absence of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 complete aneuploidy within a sample; In this case, the 22 complete chromosomal aneuploidy corresponds to the complete chromosomal aneuploidy in any one or more autosomes, and the 23rd and 24th chromosomal aneuploidy is the X chromosome. Corresponding to the complete aneuploidy of the Y chromosome Aneuploidy includes trisomy, tetrasomy, pentasomy, and other polysomy, and the number of complete chromosomal aneuploidy varies at different stages in different diseases, same disease, so different completeness that can be determined by the method of the present invention The number of chromosomal aneuploidies includes at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100. Or more chromosomal aneuploidy. According to systematic chromosomal analysis of tumors, the number of chromosomes in cancer cells ranges from hypodiploidy (much less than 46 chromosomes) to tetraploidy and more than tetraploidy (200 chromosomes). It can vary widely (see Storchova and Kuffer J Cell Sci 121: 3859-3866 [2008]). In some embodiments, the methods of the invention determine the presence or absence of more than 200 chromosomal aneuploidies in a sample from a patient suspected or known to suffer from cancer, eg, colorectal cancer. Chromosomal aneuploidy includes excess complete chromosomes including one or more chromosome loss (hypodiploidy), trisomy, tetrasomy, pentasomy and other polysomy. Excess and / or loss of chromosomal fragments can also be determined as described elsewhere herein. The method of the present invention is applied to determine the presence or absence of different aneuploidies in samples from patients suspected or known to suffer from any cancer, as described elsewhere herein. can do.

  In some embodiments, any one of chromosomes 1-22, X and Y chromosomes is present in any one or more different complete chromosomal aneuploidies in a patient test sample as described above. Can be the chromosome of interest in determining. In other embodiments, two or more chromosomes of interest are assigned to No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10, Select from any two or more of chromosomes 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, chromosomes X and Y To do. In one embodiment, any one or more chromosomes of interest selected from chromosomes 1-22, X and Y are at least 20 chromosomes selected from chromosomes 1-22, X and Y. Having chromosomes, in this case, determining the presence or absence of at least 20 different complete chromosomal aneuploidies In other embodiments, any one or more chromosomes of interest selected from chromosomes 1-22, X and Y are all of chromosomes 1-22, X and Y, and In this case, the presence or absence of complete chromosomal aneuploidy in all of chromosomes 1-22 and X and Y is determined. Different complete chromosomal aneuploidies that can be determined include complete chromosome monosomy in any one or more of chromosomes 1-22 and X and Y, and any one of X and Y. Or a complete chromosome trisomy in any one or more of the X and Y chromosomes, a complete chromosome pentasomy in any one or more of the X and Y chromosomes, and the X chromosome and There is a complete chromosomal polysomy on any one or more of the Y chromosomes.

Determination of Partial Chromosome Aneuploidy in a Patient Sample In another embodiment, the present invention provides a method for determining the presence or absence of any one or more different subchromosomal aneuploidies in a patient test sample comprising a nucleic acid molecule. provide. As the steps of the method of the present invention, (a) obtaining the sequence information of the patient nucleic acid in the test sample, and (b) using the sequence information, it was selected from chromosomes 1 to 22, the X chromosome and the Y chromosome. Identify the number of sequence tags in each of any one or more fragments in any one or more chromosomes of interest, and any one or more in any one or more chromosomes of interest Identifying the number of sequence tags of the normalized fragment sequence in each of the fragments. Normalized fragment sequences can be single fragments of chromosomes or groups of fragments from one or more different chromosomes. The method of the present invention further comprises, in step (c), the number of sequence tags identified for each of any one or more fragments in any one or more chromosomes of interest, and each normalized fragment sequence Calculate the single fragment dose for each of any one or more fragments in any one or more chromosomes of interest using the number of sequence tags identified for and optionally in step (d) Each single fragment dose in any one or more fragments in each of the one or more chromosomes of interest of any one or more chromosome fragments in any one or more of the chromosomes of interest To determine the presence or absence of any one or more different subchromosomal aneuploidies in the test sample.

  In some embodiments, step (c) comprises converting any one or more single fragment doses in any one or more chromosomes of interest into any one or more chromosomes of interest. For the number of sequence tags identified for each of any one or more fragments and the normalized fragment sequence of each of any one or more fragments in any one or more chromosomes of interest Calculated as the ratio to the number of sequence tags identified.

  In another embodiment, step (c) comprises calculating the sequence tag ratio of the fragment of interest by associating the number of sequence tags obtained for the fragment of interest with the length of the fragment of interest and The number of tags in the corresponding normalized fragment sequence is related to the length of the normalized fragment sequence, and the fragment dose of the fragment of interest as Do by calculating. Repeat this calculation for every chromosome of interest. Steps (a)-(d) can be repeated for test samples from different patients.

Means for comparing fragment doses of different sample sets can be obtained by determining a normalized segment value (NSV) that relates the fragment doses in the test sample to the average of the corresponding fragment doses in the eligible sample set. it can. NSV is calculated as follows:
here,
Are the estimated mean and standard deviation for the jth fragment dose in the eligible sample set, respectively, and x _ij is the observed jth fragment dose in test sample i.

  In some embodiments, the presence or absence of at least one subchromosomal aneuploidy is determined. In other embodiments, there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more subchromosome aneuploidies Are determined within one sample. In one embodiment, one fragment of interest selected from any one of chromosomes 1-22, X, and Y is selected from chromosomes 1-22, X, and Y. In another embodiment, two or more fragments of interest selected from chromosomes 1-22, X chromosome and Y chromosome are numbered 1, 2, 3, 4, 5, 6, 7 No. 8, No. 9, No. 10, No. 11, No. 12, No. 13, No. 14, No. 15, No. 16, No. 17, No. 18, No. 19, No. 20, No. 21, No. 22 chromosome, X chromosome And any two or more of the Y chromosomes. In one embodiment, any one or more fragments of interest selected from Chromosomes 1-22, X and Y are at least one selected from Chromosomes 1-22, X and Y, 5, 10, 15, 20, 25, 50, 75, 100 or more fragments, in this case at least 1, 5, 10, 15, 20 25, 50, 75, 100 or more different subchromosome aneuploidies. Different partial chromosome aneuploidies that can be determined include partial duplication, partial growth, partial insertion, and partial deletion.

  The sample that can be used to determine the presence or absence of chromosomal aneuploidy (partial or complete) in a patient can be any biological sample, as described elsewhere herein. The type of sample that can be used to determine aneuploidy in a patient is based on the type of disease in the patient that is known or suspected to be affected. For example, a stool sample can be selected as a DNA source to determine the presence or absence of aneuploidy associated with colorectal cancer. Preferably, the sample is a biological sample taken by non-invasive means, for example a plasma sample. As described elsewhere herein, sequencing of nucleic acids in patient samples can be performed using next generation sequencing (NGS). In some embodiments, the sequencing is massively parallel sequencing using sequencing by synthesis with a reversible dye terminator. In other embodiments, the sequencing is sequencing by ligation. In still other embodiments, the sequencing is single molecule sequencing. Optionally, an amplification step is performed prior to sequencing.

  In some embodiments, the presence or absence of aneuploidy is, for example, as described elsewhere herein, for example, lung, breast, kidney, head and neck, ovary, neck, colon, pancreas, esophagus, bladder, and Determine in patients suspected of having cancer of other organs, as well as blood cancer. Hematologic cancers include bone marrow, blood, and lymphoid cancers, which include lymph nodes, lymph vessels, condyles, thymus, spleen, and lymphatic tissues of the gastrointestinal tract. Leukemia and myeloma starting from the bone marrow and lymphoma starting from the lymphatic system are the most common types of blood cancer.

  Apparatus and system for determining CNV

  Analysis of sequencing data and diagnostics derived from this analysis are typically performed using various computer algorithms and programs. In one embodiment, the present invention provides a computer program product that generates an output indicating the presence or absence of fetal aneuploidy in a test sample. The computer product includes a computer readable medium having logic recorded in the medium to cause the processor to diagnose fetal aneuploidy, the logic calculating from at least a portion of the nucleic acid molecules from the maternal biological sample. And a computer-aided logic for analyzing fetal aneuploidy from the received data, and an output procedure for generating an output indicating the presence or type of the fetal aneuploidy.

  The methods of the invention can be performed using any CNV, for example a computer readable medium having stored thereon computer readable instructions for performing a method for identifying chromosomal aneuploidy or partial aneuploidy. Accordingly, in one embodiment, the present invention provides a computer readable medium having stored thereon computer readable instructions for performing a method for identifying complete and partial chromosomal aneuploidy, eg, fetal aneuploidy.

  The methods of the present invention can further be performed using a computer processing system configured to perform any CNV, eg, a method for identifying chromosomal aneuploidy or partial aneuploidy. Accordingly, the present invention provides a computer processing system configured to implement the above-described method. In one embodiment, the device of the invention comprises a sequencing device configured to sequence at least a portion of a nucleic acid molecule in a sample to obtain a type of sequence information as described elsewhere herein. Have.

  The invention will be described in detail in the following examples, which are not intended to limit the scope of the claims of the invention. The accompanying drawings are to be regarded as an integral part of the specification of the invention. The following examples are illustrative and do not limit the scope of the claims.

Experimental example
Example 1
Sample Processing and DNA Extraction Peripheral blood samples were taken from pregnant women who were in the first or second trimester and considered to be at risk for fetal aneuploidy. Informed consent was obtained from each party prior to blood collection. After collecting blood, amniocentesis or chorionic villi were sampled. Karyotype analysis was performed using chorionic villi or amniocentesis samples to confirm fetal karyotype.

  Peripheral blood collected from each specimen was collected in an ACD tube. One blood sample tube (6-9 mL / tube) was transferred to a 15 mL low speed centrifuge tube. The blood was centrifuged at 2640 rpm, 4 ° C. for 10 minutes using a Beckman Coulter Allegra 6R centrifuge and a rotor model GA3.8. For cell-free plasma extraction, transfer the supernatant plasma layer to a 15 mL high-speed centrifuge tube and use a Beckman Coulter Avanti J-E centrifuge and JA14 rotor to 16000 × g, 4 ° C. to 10 ° C. Centrifuge for minutes. Two centrifugation steps were performed within 72 hours after blood collection. Cell-free plasma was stored at -80 ° C and thawed only once before DNA extraction

  Cell-free DNA was extracted using Qiagen QIAmp DNA blood mini kit according to the manufacturer's instructions. 5 ml of buffer AL and 500 μl of Qiagen protease were added to 4.5 ml to 5 ml of cell-free plasma. The amount was adjusted to 10 ml with phosphate buffered saline (PBS). Using multiple columns, the precipitated cfDNA was separated from the solution by centrifugation at 8,000 RPM in a Beckman microcentrifuge. The column was washed with AW1 and AW2 buffer and cfDNA was eluted with 55 μl of nuclease-free water. About 3.5-7 ng cfDNA was extracted from plasma samples.

  All sequencing libraries were prepared from approximately 2 ng of purified cfDNA extracted from maternal plasma. Library preparation uses reagents from NEBNext® DNA Sample Prep DNA Reagent Set 1 (Part No. E6000L; New England Biolabs, Ipswich, Mass.) For Illumina® as described below. I went. Since cell-free plasma DNA is originally fragmented, no further fragmentation by spraying or sonication is performed on the plasma DNA sample. About 2 ng of the overhang portion of purified cfDNA contained in 40 μl was converted to blunt ends phosphorylated by the NEBNext® end repair module, which converted the cfDNA into NEBNext in a 1.5 ml microfuge tube. (Registered trademark) DNA sample Prep 5 μl of 10 × phosphorylated buffer, 2 μl of deoxynucleotide mixed solution (10 mM for each dNTP), 1 μl of 1: 5 diluted DNA polymerase, 1 μl of T4 DNA polymerase supplied to DNA reagent set 1 And incubated with 1 μl T4 polynucleotide kinase for 15 minutes at 20 ° C. The enzyme was then heat inactivated by incubating the reaction mixture at 75 ° C. for 5 minutes. The mixture was cooled to 4 ° C and blunt ended DNA dA tailing was performed using Klenow fragment (3'-5 'exominus) (NEBNext ™ DNA sample Prep DNA reagent set. This was done by using 10 μl of dA tailed master mix containing 1) and incubating at 37 ° C. for 15 minutes. The Klenow fragment was then heat inactivated and this inactivation was performed by incubating the reaction mixture at 75 ° C. for 5 minutes. After inactivation of the Klenow fragment, 1 μl of Illumina Genomic Adapter Oligo Mix (Part No. 1000521; Illumina Inc., Hayward, Calif.) 1: 5 dilution was used to make Illumina Adapter (Non-Index Y-Adaptors) was dA tailed by incubating the reaction mixture at 25 ° C. for 15 minutes using 4 μl of T4 DNA ligase supplied to NEBNext® DNA sample Prep DNA reagent set 1. Bound to DNA. The mixture is cooled to 4 ° C. and adapter-bound cfDNA is fed to the unbound adapter, adapter dimer, and agent-coated AMPure XP PCR purification system (Part No. A63881; Beckman Coulter Genomics, Danvers, Mass.). The purified magnetic beads were used to purify from other reagents. Perform 18 cycles of PCR to selectively enrich adapter-bound cfDNA, which is complementary to Fusion (High) Fidelity Master Mix (Finnzymes, Woburn, MA) and adapters Illumina PCR primers (Part No. 1000537 and 1000537) were used. PCR for adapter-bound DNA (98 ° C for 30 seconds; 98 ° C for 10 seconds, 65 ° C for 30 seconds and 72 ° C for 30 seconds for 18 cycles, 72 ° C for 5 minutes final extension, and In this case, the Illuminogenomic PCR primers (Part Nos. 100537 and 1000538) and fusion supplied to the NEBNext® DNA sample Prep DNA reagent set 1 according to the manufacturer's instructions. • HF • PCR master mix was used. The amplified product is purified using the purification system of Agencourt AMPure XP PCR (Agencourt Bioscience Corporation, Berverly, Mass.), Which can be obtained from the manufacturer's instructions (www.beckmangenomics.com/products/AMPureXPProtocol_000387v001 (Available in .pdf). The purified amplification product was eluted with 40 μl Qiagen EB buffer, and the concentration and size distribution of the amplified library was determined using an Agilent DNA 1000 for 2100 Bioanalyzer (Agilent technologies Inc., Santana Clara, Calif.). -Analyzed using a kit.

  The amplified DNA was sequenced using the Illumina Genome Analyzer II to obtain a 36 bp single-ended read. Only about 30 bp of random sequence information is necessary to identify sequences belonging to a specific human chromosome. Longer sequences can uniquely identify more specific targets. When sample sequencing was complete, Illumina “Sequencer Control Software” transferred the image and base call (decision) files to a Unix server running Illumina “Genome Analyzer Pipeline”. Run the Illumina “Gerald” program to align the sequence to the reference human genome, which is derived from the hg18 genome NCBI36 / hg18 defined by the National Center for Biotechnology Information (URL on the World Wide Web) ; available at http://genome.ucsc.edu/cgi-bin/hgGateway?Org=Human&db=hg18&hgsid=166260105). The sequence data generated from the above procedure for uniquely aligning with the genome was read from Gerald's output (export.txt file) by a program (c2c.pl) running on a computer running the Linnux operating system. Sequence alignments with base mismatches can only be counted when aligned uniquely to the genome and are included in the alignment count. (Replication) sequence alignments with identical starting and terminal coordination were excluded.

Less than 2 mismatches between about 5 million to about 15 million 36 bp tags were mapped to the human genome. All mapped tags were counted and included in the chromosomal dose calculation in both test and eligible samples. The region spanning from the base 0 to base 2 × 10 ⁶ , base 10 × 10 ⁶ to base 13 × 10 ⁶ , base 23 × 10 ⁶ to the end of the Y chromosome is specifically excluded from the analysis, which is the same for boys or girls. This is because tags derived from any fetus are mapped to these regions of the Y chromosome. There was some variation in the total number of sequence tags mapped to individual chromosomes between samples sequenced in the same run (running) (interchromosomal variation), with significant variation occurring in different sequencing runs (sequence sequencing). Fluctuation between Gran).

Example 2
Dose and dispersion of chromosomes 13, 18, 21, X and Y chromosomes 48 volunteer pregnant women samples to examine the degree of interchromosomal and inter-sequence variation in the number of sequence tags mapped to all chromosomes Plasma cfDNA derived from peripheral blood in was extracted, sequenced as described in Example 1, and analyzed as follows. The total number of sequence tags (sequence tag density) mapped to each chromosome was determined. Alternatively, the number of mapped sequence tags can be normalized to the length of the chromosome to generate a sequence tag density ratio. Normalization to chromosome length is not a necessary step, but can be done simply to reduce the number of digits to simplify the human interpretation. The chromosomal length that can be used to normalize the sequence tag count can be the length provided by genome.ucsc.edu/goldenPath/stats.html#hg18 on the World Wide Web.

  The sequence tag density obtained for each chromosome is related to the sequence tag density of each of the remaining chromosomes to derive a qualified chromosome dose, which is the sequence tag density of the chromosome of interest, eg, chromosome 21. And the ratio of the sequence tag density of each of the remaining chromosomes, that is, chromosomes 1 to 20, chromosome 22, and X. Table 1 shows an example of the calculated eligible chromosomal doses of interest chromosomes 13, 18, 21 and X and Y chromosomes determined in one of the eligible samples. Chromosome doses are determined for all chromosomes (Chr: chromosome) in all samples, and the average doses of chromosomes 13, 18, 21, X and Y of interest in eligible samples are shown in Table 2 and Shown in Table 3 and shown in FIGS. Figures 2-6 also show chromosomal doses for the test samples. The chromosomal dose for each chromosome of interest in the eligible sample provides a comparative measure of variation in the total number of sequence tags mapped for each chromosome of interest relative to the total number of sequence tags mapped for each remaining chromosome. Thus, a qualified chromosomal dose has a variation within the sample that most closely approximates the variation of a chromosome or chromosome group, i.e., a chromosome of interest, and serves as an ideal sequence for normalized values for further statistical evaluation. Normalized chromosomes can be identified. Figures 7 and 8 show the calculated average chromosomal doses for chromosomes 13, 18, 21 and X and Y as determined in the population of eligible samples.

  In some embodiments, the best normalized chromosome is not the least variation but has a distribution of qualified doses that most distinguishes the test sample from the qualified sample, i.e., the best normalized chromosome is not the least variation, Maximum discriminability. This discriminability is based on chromosomal dose variation and dose distribution in eligible samples.

  Tables 2 and 3 show the coefficient of variation (CV) as a measure of variability, and the Student t-test values as measures of the discriminability of chromosomes 18, 21 and X and Y. The smaller the value, the greater the discriminability. Chromosome 13 discriminability was determined as the ratio of the difference between the average chromosomal dose of the eligible sample and the chromosome 13 dose of the T13 test sample alone and the standard deviation of the average of the eligible dose.

  A qualified chromosomal dose serves as a basis for determining a threshold when identifying aneuploidy in a test sample, as described below.

  A diagnosis example of T21, T13, T18 and Turner syndrome obtained by using the discriminability of the normalized chromosome, the chromosome dose, and the chromosome of interest is described in Example 3.

Example 3
Diagnosis of fetal aneuploidy using normalized chromosomes Applying the use of chromosomal doses to biological test samples to assess aneuploidy, maternal blood test samples are taken from volunteer pregnant women and cfDNA is prepared And sequenced and analyzed as described in Examples 1 and 2.

Trisomy 21 Table 4 shows the calculated dose of chromosome 21 in an example test sample (# 11403). The calculated threshold for a positive diagnosis of T21 aneuploidy was set to a value greater than twice the standard deviation from the mean of eligible (normal) samples> (2 standard deviations). Diagnosis for T21 was made based on the fact that the chromosomal dose in the test sample was greater than the set threshold. Use chromosomes 14 and 15 as normalized chromosomes in separate calculations and use the chromosome with the lowest variability, eg, chromosome 14 or the chromosome with the highest discriminability, eg, chromosome 15 Thus, aneuploidy can be identified. Using the calculated chromosomal dose, 13 T21 samples were identified and the karyotype confirmed that the aneuploid sample was T21.

Trisomy 18 Table 5 shows the calculated dose of chromosome 18 in the test sample (# 11390). The calculated threshold for T18 aneuploidy positive diagnosis was set at twice the standard deviation (2 standard deviations) from the mean of eligible (normal) samples. Diagnosis for T18 was based on the chromosomal dose in the test sample being greater than the set threshold. Chromosome 8 was used as a normalized chromosome. In this case, chromosome 8 was the chromosome with the lowest variability or the highest discriminability. Eight T18 samples were identified using chromosomal dose and confirmed to be T18 by karyotype.

  These data indicate that normalized chromosomes have both minimum variability and maximum discriminability.

Trisomy 13 Table 6 shows the calculated dose of chromosome 13 in the test sample (# 51236). The calculated threshold for T13 aneuploidy positive diagnosis was set at twice the standard deviation (2 standard deviations) from the mean of eligible (normal) samples. Diagnosis for T13 was based on the chromosomal dose in the test sample being greater than the set threshold. Chromosome doses of chromosome 13 were calculated using chromosome 5 or a group of chromosomes 3, 4, 5, and 6 as normalized chromosomes. One T13 sample was identified.

The sequence tag density of chromosomes 3-6 is the average tag count number of chromosomes 3-6.
This data shows that the combination of chromosomes 3, 4, 5, and 6 results in lower variability than chromosome 5 and the greatest discriminability than any other chromosome. ing.

  Thus, chromosomal groups can be used as normalized chromosomes to determine chromosomal doses and identify aneuploidy.

Turner syndrome (X monosomy)
Table 7 shows the calculated doses for the X and Y chromosomes in the test sample (# 51238). The calculated threshold for a Turner syndrome (X monosomy) positive diagnosis is less than (−2 standard deviations) from the mean of eligible (normal) samples for the X chromosome and qualified for the absence of the Y chromosome (normal) ) Set smaller than (−2 standard deviation) from the average of the samples.

  Samples with X chromosome doses smaller than the set threshold chromosome dose were identified as less than one X chromosome. This same sample was determined to have less Y chromosome dose than the set threshold, indicating that the sample does not have a Y chromosome. Thus, a combination of chromosomal doses of the X and Y chromosomes was used to identify Turner syndrome (X monosomy). Therefore, according to the present invention, the CNV of a chromosome can be determined. In particular, according to the present invention, chromosomal aneuploidy is determined by massive parallel sequencing of maternal plasma cfDNA and by normalized chromosome identification for statistical analysis of sequencing data. be able to. The sensitivity and reliability of the method of the present invention can accurately perform aneuploidy tests in the first and second trimesters.

Example 4
Determination of Partial Aneuploidy Sequence dose use was applied to partial aneuploidy evaluation in biological test samples of cfDNA prepared from blood plasma and sequenced as described in Example 1. The sample was confirmed to be derived from a specimen having a partial deletion in chromosome 11 by chromosome analysis. Sequencing data analysis for partial aneuploidy (partial in chromosome 11, ie, deletion of q21-q23) was performed as described for chromosomal aneuploidy in the examples above. By mapping the sequence tag to chromosome 11 in the test sample, the tag count number obtained for the corresponding sequence of chromosome 11 in the qualified sample of the tag count number between base pairs 81000082 to 103000103 in the q-arm of the chromosome (data (Not shown) revealed a significant loss. All eligible samples using a sequence tag (810000082-103000103 bp) mapped to chromosome 11 of interest in each eligible sample and a sequence tag mapped to all 20 megabase fragments of the entire genome in the qualified sample, ie, qualified sequence tag density The qualifying sequence dose was determined as the tag density ratio in. The average sequence dose, standard deviation, and coefficient of variation are calculated for all 20 megabase fragments in the entire genome, and the 20 megabase sequence with minimal variability is the identified normalized sequence on chromosome 5 (13000014- 33000033) (see Table 8), which was used to calculate the dose of the sequence of interest in the test sample (see Table 9). Table 8 shows the dose of the sequence of interest of chromosome 11 (810000082-103000103 bp) in the test sample, which is the ratio of the sequence tag mapped to the sequence of interest and the sequence tag mapped to the identified normalized sequence. As calculated. FIG. 10 shows the sequence dose (◯) of the sequence of interest in 7 eligible samples and the sequence dose (◇) of the corresponding sequence in the test sample. The average is shown as a solid line, and the calculated threshold for partial aneuploidy positive diagnosis set to 5 standard deviations from the average is shown as a dashed line. The partial aneuploidy diagnosis was performed based on the sequence dose of the test sample that was less than the set threshold value. The test sample was verified by chromosome analysis to have a q21-q23 deletion in chromosome 11.

  Thus, in addition to identifying chromosomal aneuploidy, partial aneuploidy can be identified using the methods of the present invention.

Example 5
Demonstration of aneuploidy detection The sequence data obtained in the samples described in Examples 2 and 3 and shown in FIGS. 2-6 are further analyzed to successfully identify aneuploidy in the maternal sample. The sensitivity of the method of the present invention is shown. Normalized chromosomal doses for chromosomes 21, 18, 13, X, and Y are analyzed as a distribution (Y axis) with respect to the average standard deviation and are shown in FIG. The normalized chromosome used was indicated as a reference (denominator) (X axis).

  FIG. 11A shows the chromosomal dose distribution with respect to the standard deviation from the average of chromosome 21 in the non-abnormal sample (◯) and trisomy sample (T21; Δ) when chromosome 14 is used as the normalized chromosome of chromosome 21. Show. FIG. 11B shows the chromosomal dose distribution with respect to the standard deviation from the average of chromosome 18 in the unchanged sample (◯) and trisomy sample (T18; Δ) when chromosome 8 is used as the normalized chromosome of chromosome 18. Show. FIG. 11C uses the average sequence tag density of the group of chromosomes 3, 4, 5, and 6 as normalized chromosomes to determine the chromosomal dose of chromosome 13, and samples without change (◯) and 13 The chromosome dose distribution with respect to the standard deviation from the average of the chromosome 13 in a trisomy sample (T13; (triangle | delta)) is shown. FIG. 11D shows from the average of the X chromosome in the non-abnormal girl sample (◯), the non-abnormal boy sample (Δ), and the X monosomy sample (XO; +) when using chromosome 4 as the normalized chromosome of the X chromosome. The chromosomal dose distribution with respect to the standard deviation is shown. FIG. 11E shows that when using the average sequence tag density of the group of chromosomes 1 to 22 and the X chromosome as normalization chromosomes for determining the chromosomal dose of the Y chromosome, no abnormal boy sample (○), no abnormal girl sample (Δ) and chromosomal dose distribution with respect to the standard deviation from the average of the Y chromosome in the X monosomy sample (+).

  The data show that trisomy 21, trisomy 18 and trisomy 13 were clearly distinguished from the unaltered (normal) sample. The X monosomy sample has a clearly lower X-chromosome dose than the X-chromosome dose of the unabnormal girl sample, and a clearly lower Y-chromosome dose than the Y-chromosome dose of the unabnormal boy sample (see FIG. 11E) As easily identified. Thus, the method of the present invention is sensitive and specialized for determining the presence or absence of chromosomal aneuploidy in a maternal blood sample.

Example 6
Use massively parallel DNA sequencing on cell-free fetal DNA from maternal blood
Determination of fetal abnormalities: Test set 1 independent of training set 1
The study was approved by an institutional review board (IRB) at each institution between April 2009 and July 2010 by qualified local clinical researchers at 13 US clinical facilities. Performed under specimen protocol. Informed consent consent was obtained from each specimen prior to study involvement. The protocol was developed to obtain blood samples and clinical data to support the development of noninvasive maternal genetic diagnosis methods. Pregnant women over the age of 18 were qualified. For patients who received CVS or amniocentesis clinically, blood samples were collected prior to performing the procedure, and fetal karyotype results were collected. Peripheral blood samples (2 tubes or up to a total volume of 20 mL) were drawn from all specimens into citrate dextrose (ACD) tubes (Becton Dickinson). All samples were unidentified and assigned an anonymous patient ID number. Blood samples were transported to the laboratory overnight in a temperature controlled transport container provided for research. The time elapsed between blood draw and sample acceptance was recorded as part of the sample evaluation.

  The field study coordinator used the anonymous patient ID number to write clinical data related to the patient's current pregnancy status and history in a case report forms (CRF). Cytogenetic analysis of fetal karyotypes from samples by invasive prenatal procedures was performed at each local laboratory and the results were also recorded in the study CRF. All data obtained with the CRF was entered into a laboratory clinical database. Cell-free plasma was obtained from individual blood tubes using a two-stage centrifugation process within 24-48 hours after vascular puncture. Plasma from a single blood tube is sufficient for sequencing analysis. Cell-free DNA was extracted from cell-free plasma using the QIAmpDNA blood mini kit (Qiagen) according to the manufacturer's instructions. Cell-free DNA fragments are known to be about 170 base pairs (bp) in length (see Fan et al., Clin Chem 56: 1279-1286 [2010]), so the data before sequencing Fragmentation is not necessary.

  For the training set samples, the cfDNA is sequenced using the standard manufacturer protocol with sequencing library preparation and Illumina Genome Analyzer IIX instrument (see http://www.illumina.com/). Sent to Prognosis Biosciences (La Jolla, CA). A 36 base pair single terminal read was obtained. Collect and analyze all base calls when sequencing is complete. Sequencing libraries were prepared for test set samples and sequencing was performed on an Illumina Genome Analyzer IIX instrument. The sequencing library was prepared as follows. The full-length protocol described is substantially a standard protocol defined by Illumina and differs from the Illumina protocol only in the purification of the amplification library. Although the Illumina protocol directs gel electrophoresis to purify the amplification library, the protocol described herein uses magnetic beads for the same purification step. Approximately 2 ng of purified cfDNA extracted from maternal plasma was used to prepare the primary sequencing library, which was prepared according to the manufacturer's instructions by the Illumina NEBNext ™ DNA sample Prep DNA reagent set 1 ( Part No. E6000L; New England Biolabs, Ipswich, MA). All steps, except final purification of the adapter-bound product, performed using Agent Coat magnetic beads and reagents in place of the purification column, were performed according to the protocol according to the NENEXT ™ reagent for sample preparation of genomic DNA libraries. And sequenced using Illumina® GAII. The NEBNext (TM) protocol follows approximately that specified by Illumina (available at grcf.jhml.edu/hts/protocols/11257047_ChIP_Sample_Prep.pdf.).

  The overhang portion of approximately 2 ng of the purified cfDNA fragment contained in 40 μl is converted to blunt ends phosphorylated by the NEBNext® end repair module, which converts 40 μl of cfDNA into NEBNext®. ) 5 μl of 10 × phosphorylated buffer, 2 μl of deoxynucleotide mixed solution (10 mM for each dNTP), 1 μl of 1: 5 diluted DNA polymerase, 1 μl of T4 DNA polymerase, and 1 μl supplied to DNA sample Prep DNA reagent set 1) By culturing at 20 ° C. for 15 minutes with a T4 polynucleotide kinase. Samples were cooled to 4 ° C and purified using a QIAQuick column supplied with the QIAQuick PCR purification kit (Qiagen Valencia, CA) as follows. 50 μl reaction was transferred into a 1.5 ml microfuge tube and 250 μl Qiagen buffer PB was added. 300 μl of this result was transferred into a QIAQuick column and centrifuged at 13,000 RPM for 1 minute in a microcentrifuge tube. The column was washed with 750 μl Qiagen buffer PE and centrifuged again. Residual ethanol was removed by additional centrifugation at 13,000 RPM for 5 minutes. DNA was eluted in 39 μl Qiagen buffer EB by centrifugation. DA tailing of blunt ended DNA was performed using 16 μl of dA tailing master containing Klenow fragment (3′-5 ′ exominus) (NEBNext ™ DNA sample Prep DNA reagent set 1). The mixture was used and incubated at 37 ° C. for 30 minutes according to the manufacturer's NEBNext® dA tail-end module. Samples were cooled to 4 ° C. and purified using columns supplied with the MinElute PCR purification kit (Qiagen, Valencia, Calif.) As follows. The Klenow fragment was then heat inactivated and this inactivation was performed by incubating the reaction mixture at 75 ° C. for 5 minutes. 50 μl reaction was transferred into a 1.5 ml microfuge tube and 250 μl Qiagen buffer PB was added. 300 μl was transferred to a MinElute column and centrifuged at 13,000 RPM for 1 minute in a microcentrifuge tube. Residual ethanol was removed by additional centrifugation at 13,000 RPM for 5 minutes. DNA was eluted in 15 μl Qiagen buffer EB by centrifugation. Ten microliters of DNA eluate, along with 1 μl of Illumina Genomic Adapter Oligo Mix (Part No. 1000521) 1: 5 dilution, 15 μl of 2X quick ligation reaction buffer, and 4 μl of T4 DNA ligase, NEBNext Incubated for 15 minutes at 25 ° C. according to the (registered trademark) quick ligation module. The sample was cooled to 4 ° C. and purified using a MinElute column as follows. 150 milliliters of Qiagen buffer PE was added to 30 μl reaction, the entire volume was transferred to a MinElute column, and the column was centrifuged in a microcentrifuge tube at 13,000 RPM for 1 minute. The column was washed with 750 μl Qiagen buffer PE and centrifuged again. Residual ethanol was removed by additional centrifugation at 13,000 RPM for 5 minutes. DNA was eluted in 28 μl Qiagen buffer EB by centrifugation. 18 cycles of PCR on 23 microliters of adapter-bound DNA (98 ° C for 30 seconds; 98 ° C for 10 seconds, 65 ° C for 30 seconds and 72 ° C for 30 seconds, 18 cycles at 72 ° C) 5 min final extension and hold at 4 ° C), in this case the Illuminogenomic PCR primers (Part No.) supplied to the NEBNext® DNA sample Prep DNA reagent set 1 according to the manufacturer's instructions. 100537 and 1000538) and Fusion HF PCR Master Mix. The amplified product is purified using the purification system of Agencourt AMPure XP PCR (Agencourt Bioscience Corporation, Berverly, Mass.), Which can be obtained from the manufacturer's instructions (www.beckmangenomics.com/products/AMPureXPProtocol_000387v001 (Available in .pdf). The Agent Coat AMPure XP PCR purification system removes unincorporated dNTPs, primers, primer dimers, salts, and other contaminants, and recovers amplicons greater than 100 bp. The purified amplification product was eluted with 40 μl Qiagen EB buffer, and the concentration and size distribution of the amplified library was determined using an Agilent DNA 1000 for 2100 Bioanalyzer (Agilent technologies Inc., Santana Clara, Calif.). -Analyzed using a kit. A 36 base pair single-ended read was sequenced for both the training sample set and the test sample set.

Data analysis and sample classification Sequence reads of 36 bases in length were aligned to the human genome assembly (see http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/) obtained from the UCSC database. Alignment was performed using a Bowtie short read aligner (version 0.12.5) that allows for two base mismatches during alignment (see Langmead et al., Genome Biol 10: R25 [2009]). Only reads that were ambiguously mapped to a single genomic location were excluded. The genomic locations to which the reads were mapped were counted and included in the chromosomal dose calculation (see later description). Y chromosome region mapped without distinguishing sequence tags from boys and girls (especially from base 0 to base 2 × 10 ⁶ , base 10 × 10 ⁶ to base 13 × 10 ⁶ , base 23 × 10 ⁶ To the end of the Y chromosome) was excluded from the analysis.

  Intra-run and inter-run sequencing variations in the chromosomal distribution of sequence reads can compromise the effect of fetal aneuploidy on the mapped sequence site distribution. To correct for such variations, chromosomal doses were calculated as the number of mapping sites for a given chromosome of interest normalized to the count observed for a given normalized chromosome sequence. As described above, the normalized chromosomal sequence can be composed of a single chromosome or a chromosomal group. The normalized chromosomal sequence first considers each autosome as a potential denominator (denominator) in the ratio of counts having the chromosome of interest, and is the chromosome of interest 21, chromosome 13, chromosome 13 in the training sample set. Identified within a subset of samples that have diploid karyotypes of the X chromosome, that is, eligible samples. Denominator chromosomes, ie normalized chromosomal sequences that minimize chromosomal dose variation within and between sequencing runs, were selected. Each chromosome of interest was determined to have a well-defined normalized chromosomal sequence (denominator) (see Table 10). No single chromosome was identified as a normalized chromosomal sequence of chromosome 13, since no chromosome was determined to reduce the dose variation of chromosome 13 across the sample, ie the variance of NCV values of chromosome 13. This is because the T13 aneuploidy could not be accurately identified. Chromosomes 2-6 were selected at random and tested for ability as a group to mimic the behavior of chromosome 13. The group of chromosomes 2-6 was found to substantially reduce the dose variability of chromosome 13 in the training sample and was therefore selected as the normalized chromosome sequence for chromosome 13. As mentioned above, the variability in chromosome dose of the Y chromosome is greater than 30, and independently from these, a single chromosome was used as the normalized chromosome sequence in the Y chromosome dose determination. The group of chromosomes 2-6 was found to substantially reduce Y chromosome dose variability in the training sample and was therefore selected as the normalized chromosome sequence for the Y chromosome.

  The chromosomal dose of each chromosome of interest in the eligible sample provides a measure of variation in the total number of mapped sequence tags for each chromosome of interest relative to the total number of mapped sequence tags for each remaining chromosome. Thus, a qualifying chromosomal dose is a chromosome or chromosomal group, i.e., a normalized chromosomal sequence that provides inter-sample variation that most closely approximates the variation of the chromosome of interest and also normalizes values for further statistical evaluation. Can be identified.

  The chromosomal dose of all samples in the training set, i.e. eligible and anomalous samples, can also serve as a basis for determining the threshold when identifying aneuploidy in a test sample, as described below.

  A normalized value is determined for each chromosome of interest in each sample in the test set and used to determine the presence or absence of aneuploidy. The normalized value can be calculated as a chromosomal dose, which can be further calculated to yield a normalized chromosomal value (NCV).

  For the test set, the chromosome dose of each of the chromosome 21, chromosome 18, chromosome 13, X chromosome and Y chromosome of interest in each sample was calculated. As described in Table 10 above, the chromosome dose of chromosome 21 is the ratio of the number of tags in the test sample mapped to chromosome 21 in the test sample to the number of tags in the test sample mapped to chromosome 9. The chromosome dose of chromosome 18 is calculated as the ratio of the number of tags in the test sample mapped to chromosome 18 in the test sample and the number of tags in the test sample mapped to chromosome 8; The chromosome dose of the chromosome is calculated as the ratio of the number of tags in the test sample mapped to chromosome 13 in the test sample and the number of tags in the test sample mapped to chromosome 2-6, and the chromosome dose of X chromosome is , Map to the X chromosome in the test sample Calculated as a ratio of the number of tags in the test sample mapped to the number of tags in the test sample mapped to chromosome 6, and the chromosome dose of the Y chromosome is the number of tags in the test sample mapped to the Y chromosome in the test sample. And the ratio of the number of tags in the test sample mapped to chromosomes 2-6.

Normalized chromosome value Using the average of the chromosome dose of each chromosome of interest in each test sample and the corresponding chromosome dose determined in the eligible sample of the training set, the normalized chromosome value (NCV) is Calculated by the formula.
here,
Are the estimated mean and standard deviation for the j-th chromosome dose in the training set, respectively, and x _ij is the observed j-th chromosome dose in sample i. When the chromosomal dose is normally distributed, NCV is statistically equal to the z-score for the dose. No significant deviations from linearity in the NCV displacement-displacement value plots from the unabated samples are observed. Furthermore, NCV's normality standard test cannot reject the normality null hypothesis.

  For each test set, the NCV of each of the chromosomes of interest 21, chromosome 18, chromosome 13, X chromosome and Y chromosome in each sample was calculated. To ensure a safe and effective classification scheme, conservative boundaries were chosen for aneuploidy classification. To classify an autosomal aneuploidy state, it requires NCV> 4 to classify the chromosome as anomalous (ie, the chromosome is aneuploid), and classifies the chromosome as anomalous Required NCV <2.5. Samples with autosomes with NCV between 2.5 and 4.0 were classified as “no call”.

The classification of sex chromosomes in this test was performed by sequentially applying NCV to both the X and Y chromosomes as follows.
1. A sample was classified as a boy (XY) if NCV Y> −2.0 standard deviation from the average of the boy sample.
2. A sample was classified as a girl (XX) if NCV Y <−2.0 standard deviation from the average of the boy sample and NCV X> −2.0 standard deviation from the average of the girl sample.
3. If the average of the boy sample is NCV Y <-2.0 standard deviation and the average of the girl sample is NCV X <-3.0 standard deviation, the sample was classified as X monosomy, ie Turner syndrome. .
4). If NCV did not meet any of the above criteria, the sample was classified as “no call” for gender.

Results Demographic studies A total of 1,014 patients were enrolled from April 2009 to July 2010. Patient demographic data, invasive treatment types and karyotype results are listed in The average age of the study participants is 35.6 years (range 17-47 years) and the gestation period ranges from 1 week to 6 weeks, 1 day to 38 weeks (average is 15 weeks and 4 days) ). The overall incidence of abnormal fetal chromosome karyotype was 6.8%, of which T21 events were 2.5%. Of 946 specimens with single pregnancy and karyotype, 906 (96%) showed at least one clinically recognized risk factor for fetal aneuploidy prior to prenatal procedures. Even excluding the example of an older maternal as a single sign, the data shows a very high false positive rate for the current screening modality. Ultrasound discovery of increased nodal permeability, synovial edema, or other structural congenital anomalies most predicted abnormal karyotypes in this statistical group.

* Includes fetal results from multiple gestation periods ** Abbreviations evaluated and reported by clinicians:
AMA = Advanced Maternal Age, NT = nuchal translucency

  The distribution of the various ethnic backgrounds represented in this study population is also shown in Table 11. Of the total patients in this study, 63% were Caucasian, 17% Latin, 6% Asian, 5% mixed, and 4% African American. It was found that ethnic diversity varies greatly from place to place. For example, in one location, 60% of registrants were Latin and 26% were Caucasian specimens, and all three clinics were located in the same state and no Latin specimens were registered. As expected, no significant differences were observed for different ethnicities in the results of this study.

Training data set 1
The training set study selected 71 samples from the initial sequential accumulation of 435 samples collected from April 2009 to December 2009. All specimens harboring anomalous fetuses (abnormal karyotypes) in this primary specimen series were included in the number of specimens without anomaly for sequencing and random selection and with appropriate samples and data. The clinical characteristics of the training set patients were consistent with the demographics of the overall study shown in Table 11. The gestation period in the training set samples ranged from 10 weeks 0 days to 23 weeks 1 day. One patient with CVS below 38, amniocentesis below 32 was not identified as an invasive procedure type (no change karyotype 46, XY). 70% of patients were Caucasian, 8.5% Latin, 8.5% Asian and 8.5% mixed. Six samples that were sequenced were excluded from this set for training purposes. 4 samples from twin-bearing specimens (described further below), 1 sample with T18 contaminated during preparation, and 1 sample with fetal karyotype 69, XXX, and 65 other samples Left as a training set.

  The number of unique sequence sites (ie, tags identified as unique sites in the genome) varies from 2.2M in the early stages of the training set study to 13.7M in the late stages, which over time has improved sequencing techniques This is due to the fact that To monitor any potential shift in this 6-fold chromosomal dose at a specific site, we worked with different unaltered samples at the start and end of the study. The average number of unique sites was 3.8M and the average chromosome doses for chromosomes 21 and 18 were 0.314 and 0.528, respectively, for the first 15 non-aberrant samples. The average number of unique sites was 10.7M and the average chromosome doses for chromosomes 21 and 18 were 0.316 and 0.529, respectively, for the last 15 samples with no change. There was no statistical difference between the average chromosomal doses of chromosome 21 and chromosome 18 over the time course of the training set study.

  A training set NCV for chromosomes 21, 18, and 13 is shown in FIG. The results shown in FIG. 12 are consistent with the normality assumption that approximately 99% of diploid NCVs fall within the mean ± 2.5 standard deviations. Of this 65 sample set, 8 samples with clinical karyotype showing T21 had NCV ranging from 6-20. Four samples with clinical karyotype representing fetal T18 have NCV ranging from 3.3 to 12, and two samples with clinical karyotype representing fetal trisomy (T13) are NCV ranging from 2.6 to 4 Had. The variance of NCV in anomalous samples is due to dependence on the percentage of fetal cfDNA in individual samples.

  As with autosomes, sex chromosome means and standard deviations were established in the training set. The sex chromosome threshold could identify 100% of male and female fetuses in the training set.

Test data set 1
After establishing the average and standard deviation of chromosomal doses from the training set, a 48-sample test set was selected from a total of 575 samples collected from January 2010 to June 2010. One sample from twin pregnancies was excluded from the final analysis, leaving 47 samples as a test set. Samples prepared by personnel for sequencing and instrument operation were kept from having clinical karyotype information. The range of gestational age was similar to that seen in the training set (see Table 11). 58% of invasive procedures were CVS, higher than the overall demographics for the procedure, but similar to the training set. 50% of the specimens were Caucasian, 27% Latin, 10.4% Asian, and 6.3% African American.

In the test set, the number of specific sequence tags varied from about 13M to 26M. Chromosome doses for chromosomes 21 and 18 were 0.313 and 0.527, respectively, for the samples without anomaly. The test set NCV for chromosomes 21, 18 and 13 is shown in FIG.

* MX is a monosomy on the X chromosome with no confirmation of the Y chromosome.

  In the test set, 13/13 specimens with clinical karyotype that exhibited fetal T21 were correctly identified as having NCV in the range of 5-14. 8/8 specimens with karyotypes exhibiting fetal T18 were correctly identified as having NCVs in the range of 8.5-22. One sample with a karyotype classified as T13 in this test set had a NCV of about 3 and was classified as no call.

  For the test data set, all boy samples were correctly identified, including samples with complex karyotypes, 46, XY + marker chromosomes (not identifiable by karyotype) (see Table 3). Of the 20 girl samples, 19 were correctly identified and one girl sample was classified as no call. For 3 samples with karyotype 45, X in the test set, 2 out of 3 samples were correctly identified as X monosomy and 1 sample was classified as no call (see Table 12).

The four samples initially selected for the twin training set and one sample in the test set were from twin pregnancy. The thresholds used here can be confused by different cfDNA amounts expected in twin pregnancy settings. In the training set, the karyotype from one of the twin samples was single chorionic 47, XY + 21. The second twin sample was a dizygotic twin and amniocentesis was performed on each fetus individually. In this twin pregnancy, one of the twins was 47, XY + 21 karyotype and the other was normal karyotype 46, XX. In both cases, cell-free classification was performed based on the method described above, where the sample was classified as T21. The other two twin pregnancies in the training set were correctly classified as unchanged for T21 (all twins showed a diploid karyotype with respect to chromosome 21). For twin samples in the test set, the karyotype was established only for twin B (46, XX) and the algorithm correctly classified as no change with respect to T21.

CONCLUSION The data show that multiple abnormal fetal karyotypes can be determined from pregnant woman's blood using massively parallel sequencing. These data demonstrate that independent test set data can be used to identify a 100% accurate classification of samples with trisomy 21 and trisomy 18. Even in the case of a fetus with an abnormal chromosomal karyotype, no sample was classified incorrectly by the algorithm of the present method. Importantly, the algorithm works well in determining the presence of T21 in two sets of twin pregnancies, at least one of which is an abnormal fetus, which has not been seen before. In addition, the study screened various sequence samples from multiple centers to accurately classify pregnancy not only in the range of abnormal karyotypes likely to be encountered in commercial clinical settings, but also in common trisomy abnormalities, It shows the significance of dealing with the unacceptably high false positive rate that still remains in prenatal screening. The data provides valuable predictions for the great ability to use this method in the future. The subset analysis of the specific genomic sites shown increases with distributed consistent Poisson counting statistics.

Based on the findings of Fan and Quake, PLos One, who used massively parallel sequencing to demonstrate that the sensitivity of noninvasive prenatal decisions from maternal plasma was limited only by counting statistics (Fan and Quake, PLos One 5, e10439 [2010]). Since sequencing information has been collected across the entire genome, this method can determine any aneuploidy or other copy number polymorphisms including insertions and deletions. When the sequencing data was analyzed in a 500 kilobase bin, the karyotype from one of the samples had a small deletion between chromosomes q21 and q23 on chromosome 11 that started with q21 Was observed as a 10% reduction in the relative number of tags in Furthermore, in the training set, 3 samples had complex chromosomal karyotypes due to mosaicism in cell development analysis. These karyotypes are i) 47, XXX [9] / 45, X [6], ii) 45, X [3] / 46, XY [17], and
iii) 47, XXX [13] / 45, X [7]. Sample ii), which showed cells containing some XY, was correctly classified as XY. Samples i (from CVS treatment) and iii (from amniocentesis) show mixed XXX and X cells by cytogenetic analysis (corresponding to mosaic Turner syndrome), which are classified as no call and X monosomy, respectively. It was.

  In examining the algorithm, we observed that another interesting data point had an NCV between −5 and −6 on chromosome 21 for one sample from the test set (see FIG. 13). This sample shows diploidy on chromosome 21 by cell development, but the karyotype shows a mosaic phenomenon with partial triploidy on chromosome 9, 47, XX + 9 [9] / 46XX [6]. Indicated. Chromosome 9 is used as a denominator to determine the chromosomal dose of chromosome 21 (see Table 10), which reduces the overall NCV value. The ability to use normalized chromosomes to determine fetal trisomy 9 in this sample is demonstrated by the results obtained in Example 7 below.

  Fan's conclusion regarding the sensitivity of this method is accurate only if the algorithm used constitutes any random or systematic bias introduced by the sequencing method. If the sequencing data is not properly normalized, the resulting analysis is inferior to counting statistics. Chu et al. Describe in a recent paper that the measurements of chromosomes 18 and 13 using the massively parallel sequencing method are inaccurate and are more amenable to applying this method to the determination of T18 and T13. We concluded that further research is necessary (Chiu et al., BMJ 342: c7401 [2011]). The method used in Chu et al. Simply uses the number of sequence tags in the chromosome of interest (chromosome 21 in their case) normalized by the total number of tags in the sequencing run. The challenge of this approach is that the distribution of tags on each chromosome can fluctuate from one sequencing run to another, thus increasing the overall variability of metrics for aneuploidy determination. In order to compare the results of Chu's algorithm to the chromosomal doses used in the examples of the present invention, the test data of chromosomes 21 and 18 were used using the method recommended by Chu and others as shown in FIG. Re-analyzed. Overall, a comparison of NCV ranges for chromosomes 21 and 18 and 10/13 accurately identified from the test set according to the present invention using an NCV threshold of 4.0 for aneuploidy classification. A decrease in the determination rate of the T18 sample of the T21 sample 5/8 was observed.

  Erich et al. Only focused on T21 and used the same algorithm as Chu et al. (Ehrich et al., Am J Obstet Gynecol 204: 205 el-e11 [2011]). In addition, after observing the external reference data in their test set z-score metric, ie deviation from the training set, they retrained the test set to define the classification boundaries. In principle this approach is feasible, but in practice, how many samples need training and how often to re-establish confirmation that the classification boundary is accurate. There are challenges in deciding if you need to train. One way to alleviate this problem is to provide a control that measures the baseline and calibrates the quantitative behavior for each sequencing run.

  Data obtained using the method of the present invention show that massively parallel sequencing can determine multiple fetal chromosomal abnormalities from the plasma of pregnant women when the algorithm that normalizes the chromosome count data is optimized. The method of the present invention for quantification not only minimizes random and systematic variation between sequencing runs, but also effectively classifies aneuploidy, particularly T21 and T18, across the entire genome. be able to. More sample collection is needed to test the algorithm for T13 determination. To this end, promising, fumbling, clinical research is being conducted at multiple locations to further demonstrate the diagnostic accuracy of the present invention.

Example 7
At least five different chromosomal aneuploidies on all chromosomes of an individual test sample
Existence Determination To demonstrate the ability of the method of the invention to determine the presence or absence of any chromosomal aneuploidy in each of a set of maternal test samples (Test Set 1; Example 6), systematically determined normalized chromosomal sequences are , Identified in the unaltered sample of the training set (Training Set 1; Example 6) and used to calculate the chromosomal dose of all chromosomes in each test sample. The presence or absence of any one or more different complete fetal chromosomal aneuploidies in each sample of the test set and training set was made by sequencing information obtained from a single sequencing run for each individual sample.

  Using the chromosome density identified for each chromosome in each sample of the training set described in Example 6, i.e. the number of sequence tags, a systematically determined normalized chromosomal sequence consisting of a single chromosome or chromosome group, It was determined by calculating the single chromosome dose for chromosomes 1-22, X and Y. Systematically determined normalized chromosomal sequences for each of chromosomes 1-22, X and Y are determined by systematically calculating the chromosomal dose of each chromosome using all possible chromosome combinations as molecules. did. For example, the chromosome dose of chromosome 21 as the chromosome of interest includes (i) the number of sequence tags obtained for chromosome 21 (the chromosome of interest), and (ii) the number of sequence tags obtained for each of the remaining chromosomes. And the remaining chromosomes (excluding chromosome 21), ie all possible combinations of chromosomes from 1, 2, 3, 4, 5 etc. to 20, 21, 22, X, and Y, eg 1 + 2,1 + 3,1 + 4,1 + 5 etc. to 1 + 20,1 + 22,1 + X, and 1 + Y;, 1 + 2 + 3,1 + 2 + 4,1 + From 2 + 5 etc. to 1 + 2 + 20, 1 + 2 + 22, 1 + 2 + X, and 1 + 2 + Y; 1 + 3 + 4, 1 + 3 + 5, 1 + 3 + 6 etc. 1 from + 3 + 20, 1 + 3 + 22, 1 + 3 + X, and 1 + 3 + Y; 1 + 2 + 3 + 4, 1 + 2 + 3 + 5, 1 + 2 + 3 + 6 etc. Calculated as the sum of the number of tags obtained for + 2 + 3 + 20, 1 + 2 + 3 + 22, 1 + 2 + 3 + X, 1 + 2 + 3 + Y, etc., chromosomes 1-20 All possible combinations of chromosome 22, chromosome X, and chromosome Y are used as normalized chromosome sequences (molecules) and training sets All possible chromosomal doses for each chromosome of interest in each eligible sample in were determined. Chromosome doses in all of the training samples are determined in the same manner as chromosome 21, and the systematically determined normalized chromosome sequence for chromosome 21 is the chromosome 21 dose with the least variability across all training samples. Determined as single chromosome or chromosome group. The same analysis was repeated to determine a single chromosome or combination of chromosomes to serve as a systematically determined normalized chromosomal sequence for each of the remaining chromosomes including chromosomes 13, 18, X and Y. Using all chromosome combinations, all other chromosomes of interest in all training samples, chromosomes 1-12, chromosomes 14-17, chromosomes 19-20, chromosome 22, chromosome X and Y The normalization sequence (single chromosome or chromosome group) was determined. In this way, all chromosomes were treated as chromosomes of interest, and a systematically determined normalized sequence was determined for each of all chromosomes in each of the unaltered samples of the training set. Table 13 shows single chromosomes or chromosomal groups identified as systematically determined normalized sequences for chromosomes 1-22 and X and Y chromosomes, respectively. As is apparent from Table 13, for some chromosomes of interest, the systematically determined normalized chromosomal sequence is determined to be a single chromosome (eg, chromosome 4 is the chromosome of interest). For other chromosomes of interest, the systematically determined normalized chromosome sequence was determined to be a chromosome group (eg, when chromosome 21 is the chromosome of interest).

Table 14 shows the average, standard deviation (SD), and coefficient of variation (CV) of systematically determined normalized chromosomal sequences determined for each of all chromosomes.
Includes ^a trisomy
^b girl child

  The variance of chromosomal dose across all training samples as reflected by the CV value indicates that the use of systematically determined chromosomal sequences to obtain a large signal-to-noise ratio and dynamic range results in high aneuploidy determination as shown below Demonstrate that it can be done with sensitivity and high specificity.

  In order to demonstrate the sensitivity and specificity of the method of the present invention, the chromosomal doses of chromosomes 1-22, X and Y of interest are tested in each sample of the training set and in the test set described in Example 5. In each of the samples, the corresponding systematically determined normalized chromosomal sequence shown in Table 13 above was determined.

  Using the systematically determined normalized chromosomal sequence of each chromosome of interest, the presence or absence of any chromosomal aneuploidy was determined in each sample in the training set and in each test sample, ie, each sample is 1, 2, Complete fetuses of chromosomes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and chromosomes X and Y It was determined whether or not it contained chromosomal aneuploidy. Sequence information, i.e., sequence tag number, is obtained for all chromosomes in each sample in the training set and in each test sample, and the single chromosomal dose for each chromosome in each of the training and test samples, as described above. Calculations were made using the number of sequence tags obtained for systematically determined normalized chromosomal sequences (see Table 13) corresponding to those determined in the training set. Using the number of sequence tags obtained in each training sample for a systematically determined normalized chromosomal sequence, determine the chromosomal dose of each chromosome in each training sample, and for a systematically determined normalized chromosomal sequence The number of sequence tags obtained in each test sample was used to determine the chromosomal dose of each chromosome in each test sample. The same conservative boundaries as described in Example 6 were chosen to ensure a safe and effective aneuploidy classification.

Training Set Results Plots of chromosome doses for chromosomes 21, 18, and 13 in training set samples using systematically determined normalized chromosome sequences are shown in FIG. When using a systematically determined normalized chromosomal sequence, i.e. chromosome group 4 + 14 + 16 + 20 + 22, 8 samples with clinical karyotype showing T21 are 5.4-21. It had an NCV between 5. When using a systematically determined normalized chromosomal sequence, ie chromosome group 2 + 3 + 5 + 7, four samples with clinical karyotypes showing T18 are between 3.3 and 15.3. Had an NCV in between. When using a systematically determined normalized chromosomal sequence, ie chromosome group 4 + 5, two samples with clinical karyotype showing T13 have an NCV of 8.0 and 12.4 It was. Samples with T21 in the training set are shown as the last 8 samples (◯) of chromosome 21 data, samples with T18 in the training set are shown as the last 4 samples (Δ) of chromosome 18 data, and training set Samples having T13 in are shown as the last two samples (□) of chromosome 13 data.

  These data show that normalized chromosomal sequences can be used to determine and accurately classify different complete fetal chromosomal abnormalities. Since all samples with anomalous karyotypes have an NCV greater than 3, the probability that these samples are part of a distribution without anomalies is less than about 0.1%.

  As with autosomes, when using a systematically determined normalized chromosomal sequence for the X chromosome (ie, chromosomes 4 + 8), and also a systematically determined normalized chromosomal sequence for the Y chromosome (ie, All boys and girls fetuses in the training set were correctly identified when using chromosomes 4 + 6. In addition, all 5 samples of X monosomy were identified. FIG. 18A shows a plot of NCV determined for the X chromosome (X axis) and NCV determined for the Y chromosome (Y axis) for each sample in the training set. All samples whose karyotype is X monosomy have NCV values less than -4.83. Those X monosomy samples with karyotypes matching the 45, X karyotype (full or mosaic) have a NCV of Y close to zero as expected. The girl samples are collected around NCV = 0 for both X and Y.

Test Set Results Plots of chromosome doses for chromosomes 21, 18, and 13 in test samples using relevant systematically determined normalized chromosome sequences are shown in FIG. When using a systematically determined normalized chromosomal sequence (ie, chromosome group 4 + 14 + 16 + 20 + 22), 13 out of 13 samples with clinical karyotype showing T21 are 7 Identified with an NCV between .2 and 16.3. Systematically determined normalized chromosomal sequence (ie when using chromosome group 2 + 3 + 5 + 7), all 8 samples with clinical karyotype showing T18 are 12.7-30. Identified with an NCV between 7. When using a systematically determined normalized chromosomal sequence (ie chromosome group 4 + 5), only one sample with a clinical karyotype exhibiting T13 was identified with an NCV of 8.6. The sample with T21 in the test set is shown as the last 13 samples (◯) of the chromosome 21 data, the sample with T18 in the test set is shown as the last 8 samples (Δ) of the chromosome 18 data, and the test set The sample having T13 in is shown as the last sample (□) of chromosome 13 data.

  These data show that systematically determined normalized chromosomal sequences can be used to accurately determine and accurately classify different complete fetal chromosomal abnormalities. Similar to the training set, all samples with anomalous karyotypes have NCVs greater than 7, indicating that the probability that these samples are part of a distribution without anomalies is infinitely low ( (See FIG. 16).

  As with autosomes, when using a systematically determined normalized chromosomal sequence for the X chromosome (ie, chromosomes 4 + 8), and also a systematically determined normalized chromosomal sequence for the Y chromosome (ie, When using chromosomes 4 + 6), all male and female fetuses in the test set were correctly identified. In addition, all three samples of X monosomy were identified. FIG. 18B shows a plot of NCV determined for the X chromosome (X axis) and NCV determined for the Y chromosome (Y axis) in each sample in the test set.

  As described above, according to the method of the present invention, it is possible to determine the presence or absence of complete or partial chromosomal aneuploidy of each of chromosomes 1 to 22, chromosome X and chromosome Y in each sample. In addition to determining the complete chromosomal aneuploidy of T13, T18, T21 and X monosomy, the method of the present invention determined the presence of chromosome 9 trisomy in one of the test samples. When using a systematically determined normalized chromosomal sequence (ie 3 + 4 + 8 + 10 + 17 + 19 + 20 + 22 chromosome group), for chromosome 9 of interest, 14.4 A sample with NCV was identified (see FIG. 17). In this sample, the aneuploidy of chromosome 9 is calculated by calculating the dose of chromosome 21 (in Example 6, chromosome 9 was used as a normalized chromosome sequence for chromosome 21) to be abnormally low. Corresponded to the test sample of Example 6 which was suspected.

  This data indicates that 100% of samples with clinical karyotypes exhibiting T21, T13, T18, T9 and X monosomy were correctly identified. FIG. 19 shows NCV plots for chromosomes 1-22 in each of the 47 test samples. The median value of NCV was normalized to zero. The data show that the method of the present invention (including the use of systematically determined normalized chromosomal sequences) shows 100% sensitivity and 100% specificity for the presence of all 5 types of chromosomal aneuploidy present in this test set. And the method of the present invention clearly shows that in any sample, any complete chromosomal aneuploidy with respect to any one of chromosomes 1-22, X and Y can be identified. ing.

Example 8
Determining the presence or absence of partial fetal chromosomal aneuploidy: Determining the feline eye syndrome DiGeorge syndrome (22q11.2 deletion syndrome), a disorder caused by a deletion in chromosome 22, can lead to stunting in some body systems. Will result. Medical diseases commonly associated with DiGeorge syndrome include heart disease, immune system dysfunction, cleft palate, parathyroid dysfunction and behavioral disorders. The number and severity of disorders associated with DiGeorge syndrome vary widely. Almost all humans with DiGeorge syndrome require treatment from experts in various fields.

  To determine the presence or absence of a partial deletion of chromosome 22 in the fetus, a blood sample is taken by maternal venipuncture and cfDNA is prepared as described in the above examples. Purified cfDNA is bound to an adapter and cluster amplified using an Illumina cBot cluster station. Mass parallel sequencing is performed using a reversible dye terminator, producing millions of 36 bp reads. The sequence reads are aligned with the human hg19 reference genome and the reads uniquely mapped to the reference genome are counted as tags.

  A set of eligible samples, all known to be chromosome 22 diploids, ie a sample set of chromosomes 22 or any portion of chromosome 22 known to exist only in a diploid state. First, sequencing and analysis are performed to obtain the number of sequence tags for each of 3 megabase (MB) 1000 fragments (excluding region 22q11.2). Assuming that the human genome has about 30 billion bases (3 Gb), each of the 3 Mb 1000 fragments almost constitutes the rest of the genome. Each of the 1000 fragments is used individually or as a fragment sequence group to determine the fragment of interest, ie, the normalized fragment sequence of the 3Mb region of 22q11.2. Using the number of sequence tags individually mapped to a single 1000 bp fragment, calculate the fragment dose of the 22 Mb 11.2 3 Mb region. In addition, all possible combinations of two or more fragments are used to determine the fragment dose of the fragment of interest in all eligible samples. A single 3Mb fragment or a combination of two or more 3Mb fragments that results in a fragment with minimal variability across the sample is selected as the normalized fragment sequence.

  The number of sequence tags mapped to the fragment of interest in each eligible sample is used to determine the fragment dose in each eligible sample. The mean and standard deviation of the fragment doses in all eligible samples are calculated and used to set a threshold for comparison with the fragment doses determined in the test sample. Preferably, normalized fragment values (NSV) are calculated for all fragments of interest in all eligible samples and used for threshold setting.

  Following this, the number of tags mapped to the normalized fragment sequence in the corresponding test sample is used to determine the dose of the fragment of interest in the test sample. A normalized fragment value (NSV) is calculated for the test sample fragment, as described above, and the NCV of the fragment of interest in the test sample is compared to a threshold determined using a qualified sample, The presence or absence of 22q11.2 deletion is determined.

  A test NCV <−3 indicates that a loss of the fragment of interest, ie a partial (22q11.2) deletion of chromosome 22, is present in the test sample.

Example 9
Fecal DNA testing to predict outcome in patients with stage II colorectal cancer Approximately 30% of all stage II colorectal cancer patients will relapse and die of cancer disease. Stage II colorectal cancer in patients with relapses showed much loss on chromosomes 4, 5, 15q, 17q and 18q. In particular, the loss at 4q22.1-4q35.2 of patients with stage II colorectal cancer showed worse outcomes. Determining the presence or absence of these genomic alterations can assist patients in choosing adjuvant (adjuvant) therapy (see Brosens et al., Analytical Cellular Pathology / Cellular Oncology 33: 95-104 [2010]).

  To determine the presence or absence of one or more chromosomal deletions in the region of 4q22.1-4q35.2 of patients with stage II colorectal cancer, stool and / or plasma samples are collected from the patients. Fecal DNA was prepared according to the method described in the publication (Chen et al., J Natl Cancer Inst 97: 1124-1132 [2005]), and plasma DNA was prepared according to the method described in the above examples. DNA is sequenced according to the NGS described herein, and the patient sample sequence information is used to calculate the fragment dose of one or more fragments spanning the region 4q22.1-4q35.2. Fragment doses are determined using normalized fragment sequences that are predetermined in each set of eligible stool and / or plasma samples. The fragment dose in the test sample (patient sample) is calculated, and the presence or absence of one or more partial chromosomal deletions in the region of 4q22.1-4q35.2 determines the NSV of each fragment of interest as the eligible sample set By comparing with the threshold from NSV.

  While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that all such embodiments are merely examples. Many alterations, modifications and substitutions will occur to those skilled in the art without departing from the invention. Various alternative embodiments of the embodiments described herein can be used to practice the present invention. It is intended that the following claims define the scope of the invention and that methods, configurations, and equivalents within the scope of the claims be covered by the present invention.

Claims (32)

In a method for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in each of any four or more chromosomes of interest in a maternal test sample comprising fetal and maternal nucleic acids,
(a) obtaining sequence information regarding fetal and maternal nucleic acids in the maternal test sample using next generation sequencing (NGS);
(b) using the sequence information, identifying the number of sequence tags of each of the arbitrary four or more chromosomes of interest selected from chromosomes 1 to 22, the X chromosome and the Y chromosome; Identifying a normalized chromosome sequence tag number for each of the four or more chromosomes of interest, wherein the normalized chromosome of each of the four or more chromosomes of interest comprises normal chromosomes of interest Identified using sequence information of eligible samples taken from specimens known to be composed of cells with copy number, and:
(I) exhibit a variation in the number of sequence tags mapped to normalized chromosomes that most closely approximates the variation in the number of sequence tags mapped to the chromosome of interest;
And / or (ii) distribution of chromosomal doses of the chromosome of interest in the qualifying sample, statistically identified based on chromosomal dose variation and dose distribution with the chromosome of interest in the qualifying sample And the largest statistical difference between the distribution of chromosomal doses of the chromosome of interest in the test sample,
When,
(c) using the number of sequence tags identified for each of the any four or more chromosomes of interest and the number of sequence tags identified for the normalized chromosome, Calculating a single chromosomal dose for each of the above chromosomes of interest; and
(d) comparing each of the single chromosomal doses of each of the four or more chromosomes of interest with a threshold of each of the four or more chromosomes of interest, thereby providing any four in the maternal test sample Determining the presence or absence of different complete fetal chromosomal aneuploidies.   The method according to claim 1, wherein the step (c) includes identifying the single chromosome dose of each chromosome of interest as the number of sequence tags identified for each of the chromosomes of interest, and the normality of each of the chromosomes of interest. Calculating as a ratio to the number of sequence tags identified for the chromosomal sequence. 3. The method of claim 1 or 2, wherein step (c) comprises
(i) calculating the sequence tag density ratio for each chromosome of interest by associating the number of sequence tags identified for each chromosome of interest in step (b) with the length of each chromosome of interest Step to do,
(ii) calculating the sequence tag density ratio of each normalized chromosome by associating the number of sequence tags identified for the normalized chromosome sequence in step (b) with the length of each normalized chromosome Steps to perform, and
(iii) calculating a single chromosomal dose for each chromosome of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal dose is the subject of interest Calculating the ratio of the sequence tag density ratio in the chromosome and the sequence tag density ratio in the normalized chromosome sequence of each of the chromosomes of interest.   The method according to any one of claims 1 to 3, wherein the arbitrary four or more chromosomes of interest selected from chromosomes 1-22 and X and Y are chromosomes 1-22, A method comprising at least 20 chromosomes selected from the X and Y chromosomes and determining the presence or absence of at least 20 different complete fetal chromosomal aneuploidies.   The method according to any one of claims 1 to 3, wherein the arbitrary four or more chromosomes of interest selected from chromosomes 1-22 and X and Y are chromosomes 1-22, A method for determining the presence or absence of all different complete fetal chromosomal aneuploidies of chromosomes 1 to 22, X chromosome and Y chromosome as all X chromosome and Y chromosome.   The method according to any one of claims 1 to 5, wherein the normalized chromosome sequence is a single chromosome selected from chromosomes 1 to 22, the X chromosome, and the Y chromosome.   The method according to any one of claims 1 to 5, wherein the normalized chromosome sequence is a chromosome group selected from chromosomes 1-22 and X and Y chromosomes. In a method for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in any one or more chromosomes of interest in a maternal test sample comprising fetal and maternal nucleic acids,
(a) obtaining sequence information regarding fetal and maternal nucleic acids in the maternal test sample using next generation sequencing (NGS);
(b) Using the sequence information, identify the number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1 to 22, the X chromosome, and the Y chromosome; Identifying the number of sequence tags of the normalized fragment sequence of each of the one or more chromosomes of interest, wherein the normalized fragment sequence of each of the one or more chromosomes of interest normalizes the chromosome of interest Identified using sequence information of eligible samples taken from specimens known to be composed of cells with a large copy number and:
(I) exhibit a variation in the number of sequence tags mapped to a normalized fragment sequence that most closely approximates a variation in the number of sequence tags that map to a chromosome of interest;
And / or (ii) distribution of chromosomal doses of the chromosome of interest in the qualifying sample, statistically identified based on chromosomal dose variation and dose distribution with the chromosome of interest in the qualifying sample And the largest statistical difference between the distribution of chromosomal doses of the chromosome of interest in the test sample,
When,
(c) using the number of sequence tags identified for each of the one or more chromosomes of interest and the number of sequence tags identified for the normalized fragment sequences, Calculating a single chromosomal dose for each of the one or more chromosomes of interest; and
(d) comparing each single chromosomal dose of each of the any one or more chromosomes of interest with a threshold of each of the any one or more chromosomes of interest, thereby determining in the maternal test sample Determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies.   9. The method according to claim 8, wherein the step (c) includes identifying the single chromosome dose of each chromosome of interest as the number of sequence tags identified for each of the chromosomes of interest, and the regularity of each of the chromosomes of interest. Calculating as a ratio to the number of sequence tags identified for the fragmented sequence.   10. The method of claim 8 or 9, wherein the one or more chromosomes of interest selected from chromosomes 1-22 and X and Y are chromosomes 1-22, X and Y. A method comprising at least 20 selected chromosomes and determining the presence or absence of at least 20 different complete fetal chromosomal aneuploidies.   10. The method of claim 8 or 9, wherein said one or more chromosomes of interest selected from chromosomes 1-22 and X and Y are chromosomes 1-22, X and Y. A method for determining the presence or absence of all different complete fetal chromosome aneuploidies of chromosomes 1-22, X and Y.   12. The method according to any one of claims 1 to 11, wherein the different complete fetal chromosomal aneuploidy is selected from complete chromosomal trisomy, complete chromosomal monosomy, and complete chromosomal polysomy.   13. The method according to any one of claims 1 to 12, wherein the different complete fetal chromosomal aneuploidies are: trisomy No. 2, trisomy No. 8, trisomy No. 9, trisomy No. 21, trisomy No. 13, trisomy No. 16 , 18 trisomy, 22 trisomy, 47, XXY, 47, XXX, 47, XYY, and X monosomy.   14. The method according to any one of claims 1 to 13, wherein steps (a) to (d) are repeated for test samples from different maternal specimens, the method comprising: Determining the presence or absence of four or more different complete fetal chromosomal aneuploidies. 15. The method according to any one of claims 1 to 14, further comprising a NCV calculation step of calculating a normalized chromosome value (NCV), wherein the NCV is expressed by the following equation: Let chromosomal dose be the value that correlates to the average of the corresponding chromosomal dose in the eligible sample set,
here,

Are the estimated mean and standard deviation for each chromosome j dose in the eligible sample set, and x _ij is the observed chromosome j dose in test sample i, the NCV calculation step. In a method for determining the presence or absence of different partial fetal chromosomal aneuploidies of one or more fragments of any one or more chromosomes of interest in a maternal test sample comprising fetal and maternal nucleic acids,
(a) obtaining sequence information regarding fetal and maternal nucleic acids in the maternal test sample using next generation sequencing (NGS);
(b) a sequence tag for each of any one or more fragments in any one or more chromosomes of interest selected from chromosomes 1-22, X and Y using the sequence information; Identifying a number and identifying a sequence tag number of the normalized fragment sequence of each of any one or more fragments in the any one or more chromosomes of interest, wherein the one or The normalized fragment sequence of each of one or more fragments of a further chromosome of interest is obtained from a qualified sample taken from a specimen known to be composed of cells having a normal copy number of the chromosome of interest. Identified using sequence information and:
(I) exhibit a variation in the number of sequence tags mapped to a normalized fragment sequence that most closely approximates a variation in the number of sequence tags that map to one or more fragments of one or more chromosomes of interest ,
And / or (ii) one or more objects of interest in a qualifying sample that are statistically identified based on chromosomal dose variation and dose distribution with a chromosome of interest in a qualifying sample The largest statistic between the distribution of chromosomal doses of one or more fragments of chromosomes and the distribution of chromosomal doses of one or more fragments of one or more chromosomes of interest in the test sample To provide a scientific difference,
When,
(c) the sequence tag number identified for each of any one or more fragments in the any one or more chromosomes of interest, and the sequence tag identified for the normalized fragment sequence Calculating a single chromosomal dose for each of any one or more fragments in the any one or more chromosomes of interest using a number; and
(d) each of the single fragment doses of any one or more fragments in any one or more of the chromosomes of interest is replaced with any one of the one or more of the chromosomes of interest. Comparing to a threshold of each of one or more chromosomal fragments, thereby determining the presence or absence of any one or more different partial fetal chromosomal aneuploidies in the maternal test sample.   17. The method of claim 16, wherein step (c) comprises calculating a single fragment dose for each of any one or more chromosomal fragments in the chromosome of interest by any one or more of the chromosomes of interest. Calculated as the ratio between the number of sequence tags identified for each chromosome fragment and the number of sequence tags identified for each normalized fragment sequence of any one or more chromosome fragments in the chromosome of interest A method comprising the steps of: 18. The method according to claim 16 or 17, further comprising an NSV calculation step of calculating a normalized segment value (NSV), wherein the NSV is obtained by converting the fragment dose into a qualified sample as follows: The value associated with the average of the corresponding fragment doses in the set,

here,

Are the estimated mean and standard deviation for the jth fragment dose in the qualifying sample set, respectively, and x _ij is the observed jth fragment dose in test sample i.   19. The method according to any one of claims 16-18, wherein the different partial fetal chromosomal aneuploidy is selected from partial duplication, partial growth, partial insertion and partial deletion. .   The method according to any one of claims 16 to 19, wherein the different partial fetal chromosomal aneuploidy is a partial monosomy of chromosome 1, a partial monosomy of chromosome 4, a partial monosomy of chromosome 5, A method comprising selecting from a partial monosomy of chromosome 7, a partial monosomy of chromosome 11, a partial monosomy of chromosome 15, a partial monosomy of chromosome 17, a partial monosomy of chromosome 18, and a partial monosomy of chromosome 22.   21. The method according to any one of claims 16 to 20, wherein steps (a) to (d) are repeated for test samples from different maternal specimens, the method being different for each of the test samples. A method for determining the presence or absence of partial fetal chromosomal aneuploidy.   The method according to any one of claims 8 to 21, wherein the normalized fragment sequence is a single fragment in any one or more of chromosomes 1-22 and X and Y. .   The method according to any one of claims 8 to 21, wherein the normalized fragment sequence is a fragment group in any one or more of chromosomes 1-22 and X and Y. .   24. The method according to any one of claims 1 to 23, wherein said step (a) comprises sequencing at least a portion of said nucleic acid in said test sample, said sequence relating to said fetal and maternal nucleic acids in said test sample. How to get information.   25. The method according to any one of claims 1 to 24, wherein the test sample is a maternal sample selected from blood, plasma, serum, urine and saliva samples.   26. The method according to any one of claims 1 to 25, wherein the nucleic acid is a mixture of fetal and maternal cell-free DNA molecules.   27. The method according to any one of claims 1-26, wherein the next generation sequencing is massively parallel sequencing using sequencing by synthesis with a reversible dye terminator.   28. The method according to any one of claims 1 to 27, wherein the next generation sequencing is sequencing by ligation.   29. The method according to any one of claims 1-28, wherein the next generation sequencing comprises amplification.   30. The method according to any one of claims 1-29, wherein the next generation sequencing is a single molecule sequencing. The method according to any one of claims 1 to 7, wherein the four or more chromosomes of interest are chromosome 13, chromosome 18, chromosome 21, and chromosome X,
(I) the normalized chromosome of chromosome 13 is at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6 and chromosome 8,
(Ii) the normalized chromosome of chromosome 18 is at least one of chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.
(Iii) The normalized chromosome of chromosome 21 is at least one of chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14.
(Iv) The method, wherein the normalized chromosome of the X chromosome is at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8. Method according to any one of claims 8-15, wherein the object of interest chromosomes, chromosome 13, chromosome 18, chromosome 21, and four or more interest from the X chromosome Ru is selected A chromosome ,
(I) the normalized chromosome of chromosome 13 is selected from at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6 and chromosome 8;
(Ii) the normalized chromosome of chromosome 18 is selected from at least one of chromosome 8, chromosome 3, chromosome 5, chromosome 6, chromosome 12, chromosome 14, and chromosome 14.
(Iii) the normalized chromosome of chromosome 21 is selected from at least one of chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14;
(Iv) The method, wherein the normalized chromosome of chromosome X is selected from at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.