CN117867121A

CN117867121A - Primer combination and method for detecting tiny residues in tumor immune repertoire

Info

Publication number: CN117867121A
Application number: CN202410052339.XA
Authority: CN
Inventors: 范文涛; 潘兆东; 王勇斯; 梁浩彬; 刘冰冰; 陈辉; 吴韫里; 温韵洁; 李奎
Original assignee: Guangzhou Huayin Medical Laboratory Center Co Ltd
Current assignee: Guangzhou Huayin Medical Laboratory Center Co Ltd
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2024-04-12

Abstract

The application provides a primer combination for detecting tiny residues of a tumor immune repertoire, which can accurately detect MRD (tumor-associated digital media) of tumor patients, particularly Acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), multiple Myeloma (MM), B cell and T cell lymphoma, and the like, can detect 6 chains of the immune repertoire at one time, comprises BCR (IGH, IGK, IGL), TCR (TCR beta, TCR gamma, TCR delta), and solves the condition of missed detection of IGHD-IGHJ, IGKJ-IGKC, TRDV-TRAJ. The application further provides a kit, a detection method, computer software and a system based on the primer combination. Through UMI self correction and multiple correction of PCR amplification efficiency, nonspecific abnormal amplification and bias of amplification are avoided, and detection accuracy is ensured.

Description

Primer combination and method for detecting tiny residues in tumor immune repertoire

Technical Field

The application relates to the field of tumor detection, in particular to a primer combination for detecting tiny residues of a tumor immune group library, a kit containing the primer combination and a detection method.

Background

Minimal residual disease (Minimal Residue Disease, MRD) refers to a condition in which a patient is in clinical remission after receiving treatment, and has no clinical symptoms but tumor cells remain in the body. In order to be able to more accurately detect and dynamically monitor residual tumor cells in a patient in order to assess the effect of the treatment and predict the prognosis of the patient. At present, the research of MRD in different solid tumors is concentrated on prognosis, and the research of observation properties is favored, and the intervention research based on MRD is rarely reported. Hope to rely on the continuous development and maturity of MRD technology, while more prospective and interventional researches can be carried out in the future to test the reliability of the MRD technology, the clinical prediction value and the clinical decision reference value of the MRD are further proved.

The tumor characteristics and treatment response may vary from patient to patient. Through experimental development of the tiny residual focus, more detailed information including genetic variation, epigenetic characteristics and the like of tumor cells can be obtained, so that more accurate basis is provided for personalized treatment. By knowing the characteristics of the tiny residual lesions, doctors can better select a treatment scheme suitable for patients, and the treatment effect is improved.

The gonococcal hematological cancers mainly comprise T/B lymphocytic leukemia, lymphoma and multiple myeloma, and recent researches find that the hematological cancer recurrence is closely related to MRD.

Most T cell TCRs consist of Tcra (TRA) and Tcrp (TRB), and a few of Tcrγ (TRG) and Tcrδ (TRD), and BCR consists of two heavy chains (IGH) and two light chains (IGK, IGL).

Gene rearrangement is a rearrangement combination of different Ig or TCR fragments during lymphocyte differentiation, and its technology has great significance in early diagnosis and differential diagnosis of malignant lymphoma. Normally, the gene rearrangement of lymphocytes is random, exhibiting multiple families and polyclonality. However, during lymphogenesis, cells exhibit monoclonal proliferation, resulting in the appearance of 1 or 2 major lymphocyte clones. This monoclonal property is considered to be one of the key features of lymphomas. Thus, the gene rearrangement technique is known as a gold standard for detecting clonal proliferation of lymphocytes.

In early diagnosis and differential diagnosis of malignant lymphoma, gene rearrangement technology has been widely used. The gene rearrangement technology has better application and prospect for cases which cannot be diagnosed by conventional HE and immunohistochemical detection. This is because, in these cases, the gene rearrangement technique can more accurately detect monoclonal proliferation of lymphocytes. At the same time, gene rearrangement techniques can also help doctors to better assess the condition in those cases where malignant lymphomas have been identified.

Normal lymphocytes, in which the gene rearrangement is random without any stimulus, exhibit multiple families and polyclonalities, and have the potential to exert various cellular immune effects. In the course of lymphogenesis, under stimulation of tumor-specific antibodies or tumor-associated antigens, a specific and selective rearrangement of one or more TCR or Ig gene families of lymphocytes occurs, resulting in the monoclonal expression of TCR or Ig genes, rendering the lymphocytes clonally proliferative, resulting in the appearance of 1 or 2 major lymphocyte clones in lymph nodes, peripheral blood or bone marrow cells, which are monoclonal.

During the course of detection of recurrence of hematological tumors, traditional morphological assessment, flow cytometry, PCR, NGS, and other methodological stages have been in progress for many years. For hematological tumors, the advantages of NGS can exactly match millions or even hundreds of millions of individual B/T cell receptor genes in the immune system, thereby allowing for more accurate quantification of MRD.

Currently, detection of TCR rearrangements by two-step amplicon amplification sequencing has become a more accurate technique for assessing MRD levels. However, this method is prone to primer dimer generation, double-ended exponential amplification makes UMI ineffective for removing PCR bias, and low proportion of effective data can lead to inaccurate MRD level detection, requiring a more accurate quantification method.

Problems with the prior art also include: a reference is needed, the internal reference is unstable, and a certain deviation exists between the external reference and the internal amplification; errors caused by PCR amplification; primer dimer is relatively high; amplification is biased, and the amplification efficiency is not completely consistent, especially when a certain clone type with high relative abundance exists.

Disclosure of Invention

The application aims to formulate an MRD (multiple-strand detection device) for accurately detecting tumor patients, particularly Acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), multiple Myeloma (MM), B cell lymphoma, T cell lymphoma and the like, realize that 6 chains of an immune group library can be detected at one time, including BCR (IGH, IGK, IGL), TCR (TCRbeta, TCRgamma, TCRdelta), solve the problem of missed detection of IGHD-IGHJ, IGKJ-IGKC and TRDV-TRAJ, and avoid non-specific abnormal amplification and bias of amplification through UMI self correction and multiple correction of PCR amplification efficiency, and ensure the detection accuracy.

In order to solve the above technical problems, the present application provides a primer combination, including the following six primer sets:

a first primer set: consists of SEQ ID NO: 1-47;

second primer set: consists of SEQ ID NO: 48-59;

third primer set: consists of SEQ ID NO: 60-67;

fourth primer set: consists of SEQ ID NO: 68-98;

fifth primer set: consists of SEQ ID NO: 99-136;

sixth primer set: consists of SEQ ID NO: 137-183.

Preferably, the primer combination further comprises SEQ ID NO: 184-185.

The sequence of each primer is shown in a nucleotide or amino acid sequence table in the specification, and the occupied ratios are shown in tables 1 to 7. The ratios in tables 1 to 7 refer to the ratio of the primer to all primers in the primer set. Different primer sets are generally added to different PCR tubes for amplification, so that the relative proportions of the different primer sets are not limited.

Table 1 first primer set TRB primers.

Table 2 second primer set TRD primers.

SEQIDNO.	Primer ID	Duty ratio of	SEQIDNO.	Primer(s)ID	Duty ratio of
						48	TRD1	7.14％	54	TRD7	7.14％
49	TRD2	7.14％	55	TRD8	10％
						50	TRD3	7.14％	56	TRD9	10％
51	TRD4	7.14％	57	TRD10	10％
						52	TRD5	7.14％	58	TRD11	10％
53	TRD6	7.14％	59	TRD12	10％

Table 3 third primer set TRG primer.

Table 4 fourth primer set IGH primers.

SEQIDNO.	Primer ID	Duty ratio of	SEQIDNO.	Primer ID	Duty ratio of
						68	IGH1	1.667％	84	IGH17	1.667％
69	IGH2	1.667％	85	IGH18	1.667％
						70	IGH3	1.667％	86	IGH19	1.667％
71	IGH4	1.667％	87	IGH20	1.667％
						72	IGH5	1.667％	88	IGH21	1.667％
73	IGH6	1.667％	89	IGH22	1.667％
						74	IGH7	1.667％	90	IGH23	1.667％
75	IGH8	1.667％	91	IGH24	1.667％
						76	IGH9	1.667％	92	IGH25	1.667％
77	IGH10	1.667％	93	IGH26	1.667％
						78	IGH11	1.667％	94	IGH27	1.667％
79	IGH12	1.667％	95	IGH28	1.667％
						80	IGH13	1.667％	96	IGH29	1.667％
81	IGH14	1.667％	97	IGH30	1.667％
						82	IGH15	1.667％	98	IGH31	50％
83	IGH16	1.667％

Table 5 fifth primer set IGK primers.

Table 6 sixth primer set IGL primers.

Table 7 external standard Amt-MRD primer.

SEQIDNO.	Primer ID	Duty ratio of	SEQIDNO.	Primer ID	Duty ratio of
						184	AtMRD1-F1	50％	185	AtMRD1-R1	50％

The invention also provides a kit comprising the sequence represented by SEQ ID NO: 1-183; preferably, there is also the above-mentioned SEQ ID NO: 184-185; preferably, the primer ratios in the primer sets are shown in tables 1 to 7, for example.

The invention further provides a method for detecting tiny residues in a tumor immune group library, which is characterized by comprising the following steps:

step 1, adding the extracted DNA into a primer combination of the application for amplification, purifying a final library, and performing NGS on-machine sequencing after quality inspection to obtain sequencing data;

step 2, processing sequencing data to obtain the total number of clearready, and when the total number of clearready is smaller than 10k, re-sequencing or adding to reach the minimum standard 10k, comparing the clearready to be divided into different clonotypes, and obtaining the parameter uniqueTagCountUMI, PCRFlod, cloneCount of each clonotype;

step 3, calculating the sum sigma CloneCount of the CloneCount of each clonotype, taking CloneCount/sigma CloneCount multiplied by 100% as the relative abundance of the clonotype, and screening out 1-2 main clonotypes with the highest relative abundance;

the screening criteria were: when the sample sequencing clearready is greater than 20k, the relative abundance is more than or equal to 2.5% and more than or equal to 2×the relative abundance of the third clonotype sequenced, and the clonotype can be judged to be the main clonotype; when the sample sequencing clearready is smaller than 20K and larger than 10K, the relative abundance is more than or equal to 5% and more than or equal to 2 x the relative abundance of the third clone type can be judged to be the main clone type;

step 4, performing correction calculation to obtain MRD_UMI of the master clone type, wherein a calculation formula is as follows

MRD_UMI＝((uniqueTagCountUMI/PCRFlod)/2)/(DNAinput/gDNA)

Said MRD_UMI represents the corrected MRD level for that clonotype; the CloneCount represents the number of clones of the clonotype; the uniqueTagCountUMI represents the UMI number of the clonotype; PCRFlod is a multiple of PCR amplification efficiency and is equal to CloneCount/uniqueTagCountUMI; the DNAinput represents the input amount of sample DNA in ng; gdna=0.0064 ng.

CloneCount, uniqueTagCountUMI and the like can be directly read from the processed sequencing data. The interface of different software and the naming of the output parameters may be different, and the skilled person can find the required data in the sequencing result after understanding the meaning of the parameters.

The DNA extracted in the step 1 is divided into 6 parts on average, and each part is amplified by using one of the first primer group to the sixth primer group; the external index may or may not be added. Amplification, purification, quality control, and NGS sequencing are all routine in the art and are not additionally limited herein. The processing of the sequencing data in step 2 may use conventional data processing software in the prior art, and those skilled in the art know how to set parameters of data processing to obtain the required values such as clearready total number, which will not be described in detail herein. The screening criteria in step 3 were statistically determined by the inventors based on clinical data.

The result of the method, which is ultimately output to the physician, is 0-2 major clonotypes and MRD_UMI. Tumor patients typically have at least 1 major clonotype, and the physician can record this data and review it after each treatment or after a period of time to observe its trend. If the primary clonotype disappears, this indicates that the patient is cured; if the main clonotype and MRD_UMI are basically unchanged, the condition is relatively stable; if the major clonotype changes or the MRD_UMI changes greatly, it indicates that the tumor cells of the patient may have new variation, and the doctor needs to re-judge the disease condition in combination with other examination results to adjust the treatment method. The specific criteria for determination of tumor progression is also dependent upon the clinician's experience and other examination results, and the data measured in this application may be used as an important reference for further clinical study determination.

The present application also provides a computer program which, when executed by a processor, implements steps 2-4 of the above method.

The present application also provides a computer system comprising a memory, a processor and a computer program stored on the memory, which when executed by the processor implements steps 2-4 of the above method.

The present application also provides a computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements steps 2-4 of the above method.

Compared with the prior art, the application has the following advantages:

1. the primer design is optimized, the affinity among the primers is ensured, and the generation of primer dimer is avoided.

2. The correction of UMI is carried out by using the PCR amplification efficiency, so that the interference of inconsistent amplification efficiency is eliminated; the screening of the main clone type is matched, so that a doctor can more intuitively and accurately detect the result.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the detailed description of non-limiting embodiments, made with reference to the following drawings, in which:

FIG. 1 is a quality control diagram of the final library of example 1 prior to purification;

FIG. 2 is a quality control chart after purification using magnetic beads in example 1;

FIG. 3 is a graph showing the amplification factor distribution after 10 amplifications performed in the first round of example 1;

FIG. 4 is a schematic diagram of a computer system according to embodiment 2.

Detailed Description

For a better understanding of the present application, a more detailed description of the technical solution of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description is merely illustrative of exemplary embodiments of the application and is not intended to limit the scope of the application in any way. Like reference numerals refer to like elements throughout the specification. The expression "and/or" includes any or all combinations of one or more of the associated listed items.

It should be noted that in this specification, expressions of "first", "second", "third", etc. are used only to distinguish one feature from another feature, and do not represent any limitation on the feature. Thus, a first XXX discussed below may also be referred to as a second XXX without departing from the teachings of the present application. And vice versa.

In the drawings, the size, proportion, and shape of the drawings have been slightly adjusted for convenience of explanation. The figures are merely examples and are not drawn to scale. As used herein, the terms "about," "approximately," and similar terms are used as terms of a table approximation, not as terms of a table degree, and are intended to account for inherent deviations in measured or calculated values that will be recognized by one of ordinary skill in the art.

It will be further understood that terms such as "comprises," "comprising," "includes," "including," "having," "contains," and/or "containing" are open-ended, rather than closed-ended, terms that specify the presence of the stated features, elements, and/or components, but do not preclude the presence or addition of one or more other features, elements, components, and/or groups thereof. Furthermore, when a statement such as "at least one of the list of features" appears after the list of features, it modifies the entire list of features rather than just a single feature in the list. Furthermore, when describing embodiments of the present application, use of "may" means "one or more embodiments of the present application. Also, the term "exemplary" is intended to refer to an example or illustration.

Unless otherwise defined, all terms (including engineering and technical terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

In addition, features in the embodiments and examples of the present application may be combined with each other without conflict. In addition, unless explicitly defined or contradicted by context, the particular steps included in the methods described herein are not necessarily limited to the order described, but may be performed in any order or in parallel. The present application will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

Example 1.

And (5) preparing experiments.

The embodiment of the application uses fresh EDTA anticoagulated whole blood sample of chronic lymphocytic leukemia patient in Guangzhou hospital to extract DNA of the sample.

1. The sample is lysed.

Transferring 500-1000 mu l of anticoagulated blood into a 5-15 ml centrifuge tube, and adding 5 times volume of sterilized water into the sample. Mixing the materials for 5 to 10 times, and standing the materials for 5 minutes. White blood cells were collected by centrifugation at 2000 Xg for 5 minutes. The supernatant was carefully discarded and the residue was removed by reverse buckling on absorbent paper. Mu.l of sterile water and 25. Mu.l of proteinase K were added to the pellet and vortexed for 15 seconds to break up the pellet. Add 250. Mu.l Buffer AL to the sample and mix with high speed vortex for 15 seconds. Water bath at 65 ℃ for 30 minutes. 250 μl of absolute ethanol was added to the sample and vortexed at high speed for 15 seconds. The tube wall was collected by brief centrifugation.

DNA extraction.

The gDNA column was packed in a new collection tube. The mixture was transferred to the column. Centrifuge 10000 Xg for 1 minute. The collection tube and effluent were discarded. The gDNA column was packed in a new collection tube. Add 500. Mu.l Buffer DW1 to the column. Mix upside down for 2 times. Centrifuge 10000 Xg for 60 seconds. The effluent was discarded and the column was packed into a recovery header. 650 μl Buffer DW2 was added to the column and centrifuged at 10000 Xg for 60 seconds. The effluent was discarded and the column was packed into a recovery header. Centrifuge at 10,000Xg for 2 min. The column was transferred to a new 1.5ml centrifuge tube (self-assembly), and 100 μl of BufferAE preheated to 65 ℃ was added to the center of the membrane of the column. The mixture was left for 3 minutes. Centrifuge at 10,000Xg for 1 min. 100 μl of Bufferae preheated to 65deg.C was added to the center of the column membrane and left to stand for 3 minutes. The DNA binding column was discarded by centrifugation at 12000 Xg for 1 min. Eluted DNA was collected and the DNA sample was stored at-20 ℃. AtMRD1 was dosed according to the calculated theoretical 1% input DNA template amount. The dilution method comprises the following steps: (1) diluting AtMRD1 plasmid to 1ng/ul; (2) mixing the solution obtained in the previous step for 30s, and diluting 3ul of AtMRD1+297ul of ddH2O by 100 times; (3) mixing the solution of the previous step for 30s, and diluting 3ul of the solution of the previous step with 297ul of ddH2O for 100 times; (4) 10ul of the above solution was diluted 10-fold with 90ul of ddH2O to prepare a 1X 10-5ng/ul standard.

Primer mixing scheme: each primer upstream of each strand was mixed with the forward primer of AtMRD1 according to 1:1, mixing; each primer downstream of each strand was mixed with the reverse primer of AtMRD1 according to 1: 1. One negative quality control was added to each strand.

Step one: sequencing data is obtained.

1. One round of PCR amplification reaction.

A round of PCR amplification reactions using cDNA or DNA as templates.

(1) The following reaction systems shown in Table 8 were placed in sterilized PCR tubes:

TABLE 8 first round PCR amplification reaction System for cDNA and DNA templates.

The reverse primers for each strand were as follows: the TRB strand primer set includes: SEQ ID NO:35-47, 185; the TRD chain primer set includes: SEQ ID NO:55-59, 185; the TRG chain primer group comprises SEQ ID NO:66 67, 185; the IGH chain primer set includes: SEQ ID NO:98 185; the IGK strand primer set includes: SEQ ID NO:130-136, 185; the IGL chain primer set includes: SEQ ID NO:177-183, 185.

The primer groups are respectively added into different PCR tubes for amplification, and are not added into the same tube. The subsequent amplification in this example also follows the same procedure, i.e.the same PCR tube is assayed for only one strand (using the same primer set).

(2) The prepared reaction system was thoroughly vortexed and homogenized, and subjected to instantaneous centrifugation, followed by the PCR reaction procedure shown in Table 9 below:

TABLE 9 PCR procedure for first round PCR amplification of cDNA.

(3) After the end of the PCR procedure, the reaction product was removed, transiently centrifuged for 2s, and subjected to the next purification.

2. First round PCR product purification.

Adding magnetic beads: adding 30 mu L (1X) purified magnetic beads, mixing by vortex, standing for 5min, and removing the supernatant on a magnetic rack;

ethanol washing: washing with 200 μl of 80% ethanol for 2 times, discarding supernatant, drying at room temperature, and making precipitate with dull color;

eluting DNA: adding 22 mu L NF water, mixing, standing for 5min, placing into a magnetic rack, clarifying, collecting 20 mu L supernatant, and performing the next reaction in a new tube.

3. And (3) amplifying in a second round.

Based on the obtained pre-library, a library was prepared in the next step, and a reaction solution was prepared according to the following Table 10.

Table 10 product end repair reaction system.

The forward primers for each strand were as follows: the TRB strand primer set includes: SEQ ID NOS.1-34, 184; the TRD chain primer set includes: SEQ ID NOS.48-54, 184; the TRG chain primer group comprises SEQ ID NO.60-65, 184; the IGH chain primer set includes: SEQ ID NO.68-97, 184; the IGK strand primer set includes: SEQ ID NOS.99-129, 184; the IGL chain primer set includes: SEQ ID NO.137-176, 184.

Shaking and mixing uniformly, and reacting according to the following table program after instantaneous centrifugation.

Table 11 PCR procedure for end repair.

4. And (5) purifying magnetic beads.

Adding magnetic beads: adding 50 μl (1X) of purified magnetic beads, mixing by vortex, standing for 5min, and removing supernatant on a magnetic rack; ethanol washing: washing with 200 μl of 80% ethanol for 2 times, discarding supernatant, drying at room temperature, and making precipitate with dull color; eluting DNA: adding 22 mu L NF water, mixing, standing for 5min, placing into a magnetic rack, clarifying, collecting 20 mu L supernatant, and performing the next reaction in a new tube.

5. And (5) final library amplification.

(1) The following reaction system was set up in a sterilized PCR tube:

table 12 final library amplification reaction system.

The third primer F consisted of: the TRB strand primer set includes: SEQ ID NO:1-34, 184; the TRD chain primer set includes: SEQ ID NO:48-54, 184; the TRG strand primer set includes: SEQ ID NO:60-65, 184; the IGH chain primer set includes: SEQ ID NO:68-97, 184; the IGK strand primer set includes: SEQ ID NO:99-129, 184; the IGL chain primer set includes: SEQ ID NO:137-176, 184.

The third primer R consisted of: the TRB strand primer set includes: SEQ ID NO:35-47, 185; the TRD chain primer set includes: SEQ ID NO:55-59, 185; the TRG strand primer set includes: SEQ ID NO:66,67, 185; the IGH chain primer set includes: SEQ ID NO:98 185; the IGK strand primer set includes: SEQ ID NO:130-136, 185; the IGL chain primer set includes: SEQ ID NO:177-183, 185.

(2) The prepared reaction system is fully and uniformly vortex and centrifuged instantaneously, and the following PCR reaction procedure is carried out:

TABLE 13PCR reaction conditions.

6. And (5) final library purification and quality inspection.

Adding magnetic beads: adding 45 μl (0.9X) of purified magnetic beads, mixing by vortex, standing for 5min, and removing supernatant on a magnetic rack; ethanol washing: washing with 200 μl of 80% ethanol for 2 times, discarding supernatant, drying at room temperature, and making precipitate with dull color; eluting DNA: adding 102 mu L of NF water, mixing uniformly by vortex, standing for 5min, placing into a magnetic rack, clarifying, and taking 100 mu L of supernatant into a new 1.5mL centrifuge tube; adding magnetic beads: add 75. Mu.L (0.75X) of purified beads, vortex mix, stand for 5min, transfer supernatant to a new 1.5mL centrifuge tube on a magnetic rack. Adding magnetic beads: adding 25 μl (0.25X) of purified magnetic beads, mixing by vortex, standing for 5min, and removing supernatant on a magnetic rack; ethanol washing: washing with 200 μl of 80% ethanol for 2 times, discarding supernatant, drying at room temperature, and making precipitate with dull color; eluting DNA: adding 22 mu L of NF water, mixing uniformly by vortex, standing for 5min, placing into a magnetic rack, clarifying, and taking 20 mu L of supernatant into a new 1.5mL centrifuge tube; quality control was performed using a Qubit dsDNA HS Assay, agilent 2100 Bioanalyzer. The results of quality inspection before and after purification are shown in fig. 1 and 2, and it can be seen that good purification effect is achieved.

Ngs on-machine sequencing.

And performing NGS sequencing on the constructed immune group library DNA library.

And step two, processing sequencing data.

Sequencing to obtain original fastq data, wherein the fastq data is processed by the following steps:

(1) Performing quality control filtering by using fastp default parameters;

(2) The software cutadapt-a AGATCGGAAGAGCACACGTC-A AGATCGGAAGAGCGTCGTGT is adopted for joint removal;

(3): matching and extracting the database and UMI by using main parameters such as software mixcr align-library my_library-f-patterns hs-tag-pattern '- ((R1:)/A (UMI: N {3} AN {4} CN {3 }) (R2:)' -OsaveOriginalreads=true) and the like;

(4) Using software mixcr correctAndSortTags to correct sequencing and PCR errors within the barcode sequence;

(5) Assembling the consistency cdr sequence using software mixcr assembly;

(6) Cloning results were generated using mixcr exportClones-p full-uniqueTagCount UMI;

(7) Exogenous DNA alignment was performed using software bwa mem-t 4 and software samtools;

(8) Using script dnatarget coverage. Pl to perform exogenous DNA sequencing total coverage calculation;

table 14 quality control examples.

Samples: the sample name IGH1 is a chronic lymphocytic leukemia patient sample, and Neg-IGH is a negative control sample; total_reads, number of sequencing reads; total_bases (Gbp): sequencing the number of bases; q30_rate (%): the proportion of the base with the mass of more than Q30; successfully aligned reads qualified reads and duty cycle; atMRD1: sequence number of exogenous plasmid.

The junction quality control result shows that the data quality is high, and the analysis requirement is met. Resequencing or addition to the minimum standard of 10k is required when clearready is less than 10k.

A list of clones was obtained by the above 8 steps, and the results of selected portions are shown in Table 15, wherein, cloneId: detecting a clonotype ID number from the naming; uniqueTagCountUMI: the number of UMI sequences of the detected clonotype; clonnecount: detecting the number of all sequences of the clonotype; cloneFraction: clonotype relative abundance ratio; pcrf old: PCR amplification factors; targetSequences: detected clonotype sequences.

TABLE 15 data processing results

And step three, screening of the main clonotype.

The screening criteria were: when the sample sequencing clearready is greater than 20k, the relative abundance is more than or equal to 2.5% and more than or equal to 2×the relative abundance of the third clonotype sequenced, and the clonotype can be judged to be the main clonotype; when the sample sequencing clearready is less than 20K and greater than 10K, the relative abundance is greater than or equal to 5% and greater than or equal to 2 x the relative abundance of the third clonotype sequenced, the clonotype can be determined to be the primary clonotype.

In this example, based on the above criteria, the master clone type conforming to the above criteria was found to be CloneID 0, IGHV3-23|IGHD2-15|IGHJ4 (Table 16), the corresponding sequence was TGTGCGAAAGTCCCTGGGGGGTAGTGGTGGTAGCTGCTACTTCTG ACTACTGG, which was a minimal residual of the initial diagnosis for subsequent dynamic monitoring.

And step four, correcting and calculating MRD_UMI.

As can be seen from columns 2 and 4 of Table 16, there are certain drawbacks to the MRD levels before correction and the exogenous plasmid-calibrated MRD levels: taking the example of cloniid 0, when a large clone with high abundance is present (97.42%), the MRD level for the exogenous plasmid calibration is 2.24, and by definition of MRD is meant that the ratio of a certain class of cells in the total cells is detected and should not exceed 1, and thus the quantitative MRD value is very inaccurate when there is significant biased amplification of the clone with high abundance. Taking the example of clonneid 5859, when the number of sequences detected is 1, i.e., clonnecount is equal to 1, it is not effective in PCR amplification, and if calculated also theoretically, the detected MRD value is 3.2E-07<1E-06, which has exceeded the detection limit of NGS 1E-06.

The inventors have analyzed the above, considered that such errors are likely to be due to amplification preference, and have been experimentally verified. As shown in fig. 3: in the first round of PCR, 10 cycles exist, the theoretical PCR amplification is 10 times, and through practical statistics, the PCR amplification inevitably has amplification preference, and has the problem of amplification efficiency, as shown in fig. 3, the amplification times are as large as 2-3, the amplification times account for 53%, when obvious main clonotypes exist, the deviation is very obvious, and reaches nearly 14 times (clone number 0 in the table), the apparent quantity of AtMRD >1 is higher than that of the total cells when measured through external reference, the quantification of the high-abundance clonotypes is extremely inaccurate, and the inventor designs a correction algorithm for solving the problems, and has the following formula.

MRD_UMI＝((uniqueTagCountUMI/PCRFlod)/2)/(DNAinput/gDNA)；

The meaning of the parameters in the formula is as follows: MRD_UMI: represents the MRD level for each clonotype; UMI number representing the clonotype; PCRFlod is a multiple of PCR amplification efficiency and is equal to CloneCount/uniqueTagCountUMI; "2" in the formula means that there are 2 sets of chromosomes in each cell; DNAinput represents the sample DNA input amount (unit ng); gDNA. The DNA constant of a cell is 0.0064ng, which can be directly substituted into the calculation.

If the correction is not performed by the method of the application, the traditional calculation formula is as follows:

MRD_AtMRD＝((cloneCount/(AtCount/CountTemplate))/2/(DNAinp ut/gDNA)

the meaning of the parameters in the formula is as follows: MRD_AtMRD: represents the MRD level of the exogenous DNA; clone count per cloneCount; atCount, number of reads of exogenous DNA; countTemplate, the number of exogenous DNA templates; "2" in the formula means that there are 2 sets of chromosomes in each cell; DNAinput represents the sample DNA input amount (unit ng); gDNA. The DNA constant of a cell is 0.0064ng, which can be directly substituted into the calculation.

The calculation results are shown in table 16, wherein the MRD level_umi before correction: quantifying the level of MRD as a theoretical value; correcting MRD level_umi: MRD level corrected by PCR amplification factor; MRD level_atmrd1: MRD levels quantified by exogenous plasmids; VHits: v genes corresponding to the clone types; dhis: d gene corresponding to clone type; JHits: the J gene corresponding to the clone type.

Table 16 corrects the calculation result demonstration.

As can be seen from comparison of columns 2-4 of Table 16, the levels of MRD before correction and the levels of MRD calibrated by exogenous plasmids were inaccurate when high-abundance clonotypes and very low-abundance clonotypes were present, and the exogenous plasmids were calibrated more than 100% and were not able to reflect the clonotype's duty cycle in the cells at all. The corrected cloning type does not have the problem, and is in the middle level, the corrected MRD level and the exogenously quantified MRD level are almost in the same order of magnitude, and the corrected MRD quantification has excellent quantification capability.

Example 2.

The application also provides a computer system for detecting the tiny residues of the tumor immune group library, which can be realized in the forms of a mobile terminal, a Personal Computer (PC), a tablet personal computer, a server and the like. Referring now to FIG. 4, a schematic diagram of a primer combination system suitable for use in implementing embodiments of the present application for detecting microscopic residues of tumor immunity repertoires is shown.

As shown in fig. 4, the computer system 300 includes one or more processors, communication sections, etc., such as: one or more Central Processing Units (CPUs) 301, and/or one or more image processors (GPUs) 313, etc., which may perform various suitable actions and processes based on executable instructions stored in a Read Only Memory (ROM) 302 or loaded from a storage 308 into a Random Access Memory (RAM) 303. The communications portion 312 may include, but is not limited to, a network card, which may include, but is not limited to, a IB (Infiniband) network card.

The processor may communicate with the rom 302 and/or the ram 303 to execute executable instructions, and is connected to the communication unit 312 through the bus 304, and communicates with other target devices through the communication unit 312, so as to perform operations corresponding to any of the methods provided in the embodiments of the present application, for example: steps two to four in example 1. In addition, in the RAM 303, various programs and data required for device operation can also be stored. The CPU 301, ROM 302, and RAM 303 are connected to each other through a bus 304. In the case of RAM 303, ROM 302 is an optional module. The RAM 303 stores executable instructions that cause the processor 301 to execute operations corresponding to the communication methods described above, or write executable instructions to the ROM 302 at the time of execution. An input/output interface (I/O interface) 305 is also connected to the bus 304. The communication unit 312 may be integrally provided or may be provided with a plurality of sub-modules (e.g., a plurality of IB network cards) and be connected to a bus link.

The following components are connected to the I/O interface 305: an input section 306 including a keyboard, a mouse, and the like; an output portion 307 including a Cathode Ray Tube (CRT), a Liquid Crystal Display (LCD), and the like, and a speaker, and the like; a storage section 308 including a hard disk or the like; and a communication section 309 including a network interface card such as a LAN card, a modem, or the like. The communication section 309 performs communication processing via a network such as the internet. The drive 310 is also connected to the I/O interface 305 as needed. Removable media 311, such as magnetic disks, optical disks, magneto-optical disks, semiconductor memory, and the like, is mounted on drive 310 as needed.

It should be noted that the architecture shown in fig. 4 is only an alternative implementation, and in a specific practical process, the number and types of components in fig. 4 may be selected, deleted, added or replaced according to actual needs; in the setting of different functional components, implementation manners such as separate setting or integrated setting may be adopted, for example, the GPU and the CPU may be separately set or the GPU may be integrated on the CPU, the communication portion 312 may be separately set, may be integrally set on the CPU or the GPU, and the like. Such alternative embodiments fall within the scope of the present disclosure.

In particular, the process described with reference to flowchart 4 may be implemented as a computer program product according to the present application. For example, the present application provides a computer program product comprising computer readable instructions which, when executed by a processor, perform the following: steps two to four in example 1. In such embodiments, the computer program product may be downloaded and installed from a network via the communication portion 309 and/or read and installed from the removable medium 311. The above-described functions defined in the method of the present application are performed when the computer program product is executed by a Central Processing Unit (CPU) 301.

The technical solutions of the present application may be implemented in many ways. For example, the techniques of this application may be implemented by software, hardware, firmware, or any combination of software, hardware, and firmware. The order of steps used to describe the method is provided only for the purpose of more clearly describing the technical solution. The method steps of the present application are not limited to the order specifically described above unless specifically limited. Furthermore, in some embodiments, the present application may also be implemented as a storage medium storing a computer program product.

The above description is merely illustrative of the implementations of the application and of the principles of the technology applied. It should be understood by those skilled in the art that the scope of protection referred to in this application is not limited to the specific combination of the above technical features, but also encompasses other technical solutions formed by any combination of the above technical features or their equivalents without departing from the technical concept. Such as the above-described features and technical features having similar functions (but not limited to) disclosed in the present application are replaced with each other.

Claims

1. A primer combination comprising the following six primer sets:

a first primer set: consists of SEQ ID NO: 1-47;

second primer set: consists of SEQ ID NO: 48-59;

third primer set: consists of SEQ ID NO: 60-67;

fourth primer set: consists of SEQ ID NO: 68-98;

fifth primer set: consists of SEQ ID NO: 99-136;

sixth primer set: consists of SEQ ID NO: 137-183.

2. The primer combination of claim 1, wherein the primer combination further comprises SEQ ID NO:184-185, the two external primers each being 50%.

3. The primer combination according to claim 1, wherein,

in the first primer set, SEQ ID NO:1-34 is 1.47% of each primer as set forth in SEQ ID NO:35-47 at 3.85% of each primer;

in the second primer set, SEQ ID NO:48-54 is 7.14% of each primer, said SEQ ID NO:55-59 was 10% of each primer;

in the third primer set, SEQ ID NO:60-65 is 8.33% of each primer, said SEQ ID NO:66-67 was 25% of each primer;

in the fourth primer set, SEQ ID NO:68-97 is 1.67% of each primer, said SEQ ID NO:98 was 50% of the primer;

in the fifth primer set, SEQ ID NO:99-129 is 1.61% of each primer set forth in SEQ ID NO:130-136 was 7.14% of each primer;

in the sixth primer set, SEQ ID NO:137-176 is 1.25% of each primer, said SEQ ID NO:177-183 was 3.57% of each of the primers.

4. A kit comprising the primer combination of any one of claims 1-3.

5. A method for detecting microscopic residues in a tumor immune repertoire, comprising the steps of:

step 1, adding the extracted DNA into the primer combination shown in any one of claims 1-3 for amplification, purifying a final library, and performing NGS on-machine sequencing after quality inspection to obtain sequencing data;

MRD_UMI＝((uniqueTagCountUMI/PCRFlod)/2)/(DNAinput/gDNA)

Said MRD_UMI represents the corrected MRD level for that clonotype; the CloneCount represents the number of clones of the clonotype; the uniqueTagCountUMI represents the UMI number of the clonotype; PCRFlod is the multiple of PCR amplification efficiency; the DNAinput represents the input amount of sample DNA in ng; gdna=0.0064 ng.

6. The method according to claim 5, wherein in step 1, the extracted DNA is divided into 6 parts in average, and each part is amplified using one of the first primer set to the sixth primer set.

7. The method of claim 6, wherein each of the sequences further uses SEQ ID NO: 184-185.

8. A computer program, which, when being executed by a processor, implements steps 2-4 of the method of claim 5.

9. A computer system comprising a memory, a processor and a computer program stored on the memory, characterized in that the computer program, when executed by the processor, implements steps 2-4 of the method of claim 5.

10. A computer readable storage medium, on which a computer program is stored, characterized in that the computer program, when being executed by a processor, carries out the steps 2-4 of the method of claim 5.