CN108148900A - Sequencing approach, kit and its application of sequencing mistake are reduced based on molecular label and the sequencing of two generations - Google Patents
Sequencing approach, kit and its application of sequencing mistake are reduced based on molecular label and the sequencing of two generations Download PDFInfo
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Abstract
The invention discloses a kind of sequencing approaches, kit and its application, sequencing approach that sequencing mistake is reduced based on molecular label and the sequencing of two generations to include：S1. 4 16 kinds are synthesized containing the normal chain P5 of connector of different fixed distinguished sequence labels and anti-chain P7 for each sample, P5 and P7 is annealed, forms Y-shaped connector；S2. by template DNA end-filling, and in 3 ' end addition A bases；S3. by the Y-shaped connector template DNA both ends that are connected to that treated；S4. the template DNA of recycling connection top connection, and Enrichment Amplification is carried out to template DNA with the adapter-primer with sample label；S5. the product of enriched amplification is recycled；S6. the sequencing of two generations is carried out to Enrichment Amplification product.The method of the present invention can effectively identify the mutation in double-stranded DNA primary template, especially asymmetric mutation, it thus can effectively identify the amplification mistake introduced since first time Enrichment Amplification, Illumina is single-ended and both-end sample label sequencing pattern under, by the fixation distinguished sequence of 4 16 kinds of exclusive connectors of sample, sample label drifting problem can be effectively solved.
Description
Technical field
The present invention relates to biotechnologies more particularly to a kind of be sequenced based on molecular label and two generations to reduce sequencing mistake
Sequencing approach, kit and its application.
Background technology
The sequencing of two generations connects target template DNA top connection sequence, and obtain more by Enrichment Amplification by library construction
Then the library built is carried out deep sequencing by more target template DNAs in two generation microarray datasets.
Archaeal dna polymerase is all used when library construction and the platform sequencing of more than microarray dataset, to ensure the accuracy of detection,
Using high-fidelity DNA polymerase, the fidelity of such archaeal dna polymerase is higher than 10-9, i.e., often synthesize 109It can be random during a base
There is the mistake of 1 base.Therefore, the sequencing mistake about 0.1% of the technology.
Therefore, it when more than technology is used to carry out the sequencing of low frequency nucleotide variation Related product, is influenced by sequencing mistake
It can not effectively identify true low frequency abrupt information.For example, tumour liquid biopsy needs to detect one thousandth even a ten thousandth
Low frequency is mutated；The sample progress population that the sequencing analysis of microbial diversity needs to reach Monopterus albus thousands of or even up to ten thousand is more
Sample is analyzed or the diversity of functional gene is analyzed, and needs to differentiate the one thousandth even base difference of a ten thousandth.
When technology sequencing mistake reaches 0.1%, true mutation is submerged in the pseudomutation of sequencing mistake and None- identified.
Mistake is sequenced to reduce, Swift Biosciences companies provide solution, i.e. Accel-NGS 2S
Indexed Adapters.In library construction, the random sequence that 9 bases are added in the P5 ends label position of connector is marked
Label, normal chain and minus strand to primary template are marked respectively.It, can be more than when analyzing two generation sequencing results
The sequence of 9 bases identifies the normal chain of primary template and minus strand respectively, and repetitive sequence is merged, while school each other
It tests.The mistake brought on primary template by Enrichment Amplification and sequencing can detected and correct, and sequencing mistake is reduced with this.
Meanwhile Integrated Device Technology, Inc. also provides solution, i.e.,Dual Index UMI Adapters.In library
During structure, the random sequence in addition increasing by 9 bases before the P7 ends label of connector makees label, to the normal chain of primary template and negative
Chain is marked respectively.It, can be by the sequences of above 9 bases by primary template when analyzing two generation sequencing results
Normal chain and minus strand identify respectively, repetitive sequence is merged, while verifies each other.Expanded on primary template by enrichment
The mistake that increasing and sequencing are brought can detected and correct, and sequencing mistake is reduced with this.
More than technology adds the random sequence of 9 bases to reduce sequencing mistake by the label position of P5 or P7 in connector
Accidentally, but the normal chain due to the primary template from same double-stranded DNA and minus strand are with different labels, and the tag combination is not
It determines, therefore, it is impossible to by the matched reduction one by one of the normal chain from same double-stranded DNA primary template and minus strand.Therefore, it is impossible to
Identify the asymmetric mutation in double-stranded DNA primary template.
Meanwhile in the first round amplification of the Enrichment Amplification after connecting the connector with label, since archaeal dna polymerase exists
The amplification mistake of 10e-9, the mistake can cause the amplified production of the normal chain/minus strand primary template exist it is asymmetric be mutated, with
The random sequence of 9 bases of the label position of P5 or P7 is come when reducing sequencing mistake, normal chain/minus strand has 2 in errors present
Middle base can not be effectively differentiated to mistake.
When Illumina microarray datasets carry out the sequencing of two generations, if being carried out at the same time multiple samples, sample label can occur
Sequence drifting problem, that is, the label for becoming No. 2 or other samples of the sample label mistake of No. 1 sample causes other samples
Middle having carried for mistake is not belonging to its sequence, therefore, the abrupt information of introducing that can be wrong.Swift Biosciences are public
The method of department and Integrated Device Technology, Inc. is both-end sample label, under both-end sample label sequencing pattern, can effectively correct sample mark
Label drift, but under single-ended sample label sequencing pattern, then can not solve the problems, such as this.
Invention content
For above-mentioned technical problem, sequencing mistake is reduced based on molecular label and the sequencing of two generations the present invention provides a kind of
Sequencing approach, kit and its application, wherein, the sequencing approach includes the following steps：
S1. for different samples, synthesis 4-16 kinds contain the normal chain P5 of the connector of different fixed distinguished sequence labels and anti-
Chain P7 anneals the P5 of various terminal and P7, forms 4-16 kind Y-shaped connectors；
S2. template DNA is subjected to end-filling, and 3 ' the end addition A bases after filling-in；
S3. 4-16 kind Y-shaped connectors are connected to step S2 treated the both ends of template DNA；
S4. the template DNA of recycling connection top connection, and with the adapter-primer with sample label to the template DNA of recycling
Carry out Enrichment Amplification；
S5. the product through step S4 Enrichment Amplifications is recycled；
S6. the sequencing of two generations is carried out to Enrichment Amplification product.
The sequencing approach, wherein, the fixed distinguished sequence label is located at the 3 ' ends of P5, the fixed distinguished sequence
The reverse complementary sequence of label is located at the 5 ' ends of P7.
The sequencing approach, wherein, the connector that the 4-16 kinds contain different fixed distinguished sequence labels is equimolecular
Concentration mixes, wherein, the fixed distinguished sequence label is by 2-10 base composition.
The sequencing approach, each sample, which contains specific 4-16 kinds difference, fixes specially sequence label, each sample
Between fixation specially sequence label is different.
The sequencing approach, wherein, P5 is the deoxynucleotide sequence as shown in SEQ ID NO.1, and the P7 is such as
Deoxynucleotide sequence shown in SEQ ID NO.2.
The sequencing approach, wherein, include forward primer and anti-with sample label adapter-primer in the step S4
To primer, the forward primer is the deoxynucleotide sequence as shown in SEQ ID NO.3, and the reverse primer is such as SEQ ID
Deoxynucleotide sequence shown in NO.4.
The present invention also provides a kind of kit for realizing sequencing approach as described above, including connector, connector connection examination
Agent, adapter-primer, the nucleic acid enriching magnetic bead for expanding connection product.
The kit, wherein, the connector is formed for P5 sequences and P7 sequence anneals, and the P5 is such as SEQ ID
Deoxynucleotide sequence shown in NO.1, the P7 are the deoxynucleotide sequence as shown in SEQ ID NO.2.
The kit, wherein, expand connection product adapter-primer include forward primer and reverse primer, it is described just
It is the deoxynucleotide sequence as shown in SEQ ID NO.3 to primer, the reverse primer is de- as shown in SEQ ID NO.4
Oxygen nucleotide sequence.
The present invention also provides the application of sequencing approach as described above, wherein, the sequencing approach can individually or with spy
Needle capture technique be conjointly employed in detection in Gene Mutation or applied to microbial diversity sequencing, population diversity analysis or
The diversity analysis of functional gene.
Relative to the prior art, the embodiment of the present invention has the following advantages:
1st, the present invention can carry out the testing result of the normal chain from same double-stranded DNA primary template and minus strand one by one
Matching, it is possible thereby to effectively identify the mutation in double-stranded DNA primary template, especially asymmetric mutation.
2nd, the amplification mistake introduced since first time Enrichment Amplification can be effectively identified.
3rd, Illumina is single-ended and both-end sample label sequencing pattern under, pass through consolidating for the exclusive 4-16 kind connectors of sample
Determine distinguished sequence, can effectively solve the problems, such as that sample label is drifted about.
Description of the drawings
Fig. 1 is the schematic diagram that the connector containing molecular label is connected to template DNA in the embodiment of the present invention.
Specific embodiment
Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, the every other implementation that those skilled in the art are obtained without creative efforts
Example, shall fall within the protection scope of the present invention.
The present invention provides it is a kind of based on molecular label and two generations sequencing reduce sequencing mistake sequencing approach, kit and
It is applied.
Wherein, the construction method of the sequencing approach of sequencing mistake is reduced based on molecular label technology and two generation PCR sequencing PCRs：It is first
First, at 3 ' ends of connector P5 chains plus the fixation distinguished sequence of 2-10 bases at 5 ' ends of connector P7 chains spy is fixed plus the former
Different sequence reverse complemental terminal sequence.The connector of each reverse complemental is individually annealed, is formed and fixes special sequence with 2-10 bases
The Y-shaped connector of row.Secondly, when carrying out library construction, 4-16 kind connector equimolecular quantity concentration is mixed, each sample
Originally it is respectively provided with unique 4-16 kinds connector.Finally, when two generation sequencing results are analyzed, it is according to unique fixed distinguished sequence
Label merges the repetition of the normal chain of primary template or minus strand, correction Enrichment Amplification and sequencing mistake；Same double-strand will be come from
The normal chain and minus strand of DNA merges, correction Enrichment Amplification and sequencing mistake.Since each sample is respectively provided with unique connector, because
This, under the sequencing pattern of single-ended sample label, it is possibility to have the problem of effect correction sample label is drifted about.
Specifically, the sequencing approach includes the following steps：
S1. for different samples, synthesis 4-16 kinds contain the normal chain P5 of the connector of different fixed distinguished sequence labels and anti-
Chain P7 anneals the P5 of various terminal and P7, forms 4-16 kind Y-shaped connectors.
Added at 3 ' ends of connector P5 chains plus the fixation distinguished sequence label of 2-10 bases at 5 ' ends of connector P7 chains
The former distinguished sequence reverse complemental terminal sequence.The connector of each reverse complemental is individually annealed, formation is fixed with 2-10 bases
The Y-shaped connector of distinguished sequence.
Needle is for each sample, synthesis 4-16 kinds contain the connector of different fixed distinguished sequence labels, by subsequently connecting shape
Into the template DNA with fixed distinguished sequence labels different in 4-16；The fixation distinguished sequence mark used without same sample
The connector of label is different, is convenient for being carried out at the same time the sequencing of different samples in this way.
Wherein, normal chain P5 is the deoxynucleotide sequence as shown in SEQ ID NO.1, and anti-chain P7 is such as SEQ ID NO.2
Shown deoxynucleotide sequence, particular sequence are as follows：
P5：ACACTCTTTCCCTACACGACGCTCTTCCGATCT[NNNNNNNNNN]-s-T；P7：P-
[NNNNNNNNNN]AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC.Wherein, x is one in four kinds of bases A, T, C and G
Kind；The T at the 3 ' ends of P5 carries thio-modification；And 5 ' the ends of P7 have carried out phosphorylation modification.
S2. template DNA is subjected to end-filling, and 3 ' the end addition A bases after filling-in；
S3. 4-16 kind Y-shaped connectors are connected to step S2 treated the both ends of template DNA；
Preferably, the connector that the 4-16 kinds contain different fixed distinguished sequence labels is the mixing of equimolecular concentration.Exist
When carrying out library construction, 4-16 kind connector equimolecular quantity concentration is mixed, each sample is made to be respectively provided with unique 4-
16 kinds of connectors.
S4. with the template DNA of nucleic acid enriching magnetic bead recycling connection top connection, remove remaining not connected connector, enzyme, salt from
Sub and other organic reagents carry out Enrichment Amplification with the adapter-primer with sample label to the template DNA of recycling later；
The adapter-primer with sample label includes forward primer and reverse primer, the forward primer in the step S4
For the deoxynucleotide sequence as shown in SEQ ID NO.3, the reverse primer is the deoxyribonucleoside as shown in SEQ ID NO.4
Acid sequence, particular sequence are as follows：
Forward primer：
CAAGCAGAAGACGGCATACGAGAT[NNNNNNNN]GTGACTGGAGTTCAGACGTG-s-T；
Reverse primer：
AATGATACGGCGACCACCGAGATCTACAC[NNNNNNNN]ACACTCTTTCCCTACACGA-s-C。
Wherein, N is one kind in four kinds of bases A, T, C and G.
Also, 3 ' end T of the forward primer carry thio-modification.
Wherein, the NNNNNNNN sequences in forward primer are sample label sequence, and the NNNNNNNN in reverse primer is just
The reverse complementary sequence of sample label sequence into primer.
3 ' C-terminals of the reverse primer carry thio-modification.
S5. the product through step S4 Enrichment Amplifications, removal primer dimer, enzyme, salt ion are recycled with nucleic acid enriching magnetic bead
And other organic reagents.；
In the present embodiment, more than Enrichment Amplification product is recycled using nucleic acid enriching magnetic bead, this recovery method is efficient, fast
Degree is fast, can remove primer dimer, enzyme, salt ion and other impurities.
S6. the sequencing of two generations is carried out to Enrichment Amplification product.
Wherein, the sequencing equipment that two generations PCR sequencing PCR of the present invention uses include but not limited to Roche/454,
Illumina sequenators (Novaseq series, NextSeq series, Hiseq series, MiSeq series, X-Ten, X-Five and
Subsequently with principle sequenator series), sequenator, the LifeTech of BGI (Hua Da company, BGI500 series and follow-up sequenator)
Instrument (Ion, Proton and follow-up sequencing instrument series) is sequenced.
The present invention also provides the kits for realizing sequencing approach as described above, including connector, connector connection reagent, expand
Increase adapter-primer, the nucleic acid enriching magnetic bead of connection product.
Wherein, the connector is formed for P5 sequences and P7 sequence anneals, and the P5 is the deoxidation as shown in SEQ ID NO.1
Nucleotide sequence, the P7 are the deoxynucleotide sequence as shown in SEQ ID NO.2.
The adapter-primer for expanding connection product includes forward primer and reverse primer, and the forward primer is such as SEQ ID
Deoxynucleotide sequence shown in NO.3, the reverse primer are the deoxynucleotide sequence as shown in SEQ ID NO.4.
The present invention also provides the application of sequencing approach as described above, wherein, the sequencing approach can individually or with spy
Needle capture technique be conjointly employed in detection in Gene Mutation or applied to microbial diversity sequencing, population diversity analysis or
The diversity analysis of functional gene.
The experimental method that concrete operations in the present invention are provided with reference to the product description accordingly used, does not do in detail herein
It states.
Verification test
The present invention is verified using standard items NA12878 and NA24875, uses KAPA Hyperplus DNA
Library Kit carry out library construction, and carry out the sequencing of single-ended sample label in Illumina platforms.
Analysis of biological information the results show that the present invention can effectively by the normal chain from same double-stranded DNA template and
Minus strand matches, and corrects its amplification mistake and sequencing mistake；It can effectively identify single-ended sample in Illumina microarray datasets
Sample label drifting problem under label sequencing pattern.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
The present invention is described in detail, it will be understood by those of ordinary skill in the art that：It still can be to aforementioned each implementation
Technical solution recorded in example modifies or carries out equivalent replacement to which part technical characteristic；And these modification or
It replaces, the spirit and scope for various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution.
Sequence table
<110>Shenzhen is because closing bio tech ltd
<120>Sequencing approach, kit and its application of sequencing mistake are reduced based on molecular label and the sequencing of two generations
<130> HZ1810064-SZW
<141> 2018-01-24
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
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<213>Artificial synthesized sequence (Synthetic sequence)
<220>
<221> misc_feature
<222> (34)..(43)
<223> n=a,t,c or g.
<400> 1
acactctttc cctacacgac gctcttccga tctnnnnnnn nnnt 44
<210> 2
<211> 44
<212> DNA
<213>Artificial synthesized sequence (Synthetic sequence)
<220>
<221> misc_feature
<222> (1)..(10)
<223> n=a,t,c or g.
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nnnnnnnnnn agatcggaag agcacacgtc tgaactccag tcac 44
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<213>Artificial synthesized sequence (Synthetic sequence)
<220>
<221> misc_feature
<222> (25)..(32)
<223> n=a,t,c or g.
<400> 3
caagcagaag acggcatacg agatnnnnnn nngtgactgg agttcagacg tgt 53
<210> 4
<211> 57
<212> DNA
<213>Artificial synthesized sequence (Synthetic sequence)
<220>
<221> misc_feature
<222> (30)..(37)
<223> n=a,t,c or g.
<400> 4
aatgatacgg cgaccaccga gatctacacn nnnnnnnaca ctctttccct acacgac 57
Claims (10)
1. a kind of reduce the sequencing approach of sequencing mistake based on molecular label and the sequencing of two generations, which is characterized in that the sequencing side
Method includes the following steps：
S1. for different samples, synthesis 4-16 kinds contain the normal chain P5 of connector of different fixed distinguished sequence labels and anti-chain P7,
The P5 of various terminal and P7 are annealed, form 4-16 kind Y-shaped connectors；
S2. template DNA is subjected to end-filling, and 3 ' the end addition A bases after filling-in；
S3. 4-16 kind Y-shaped connectors are connected to step S2 treated the both ends of template DNA；
S4. the template DNA of recycling connection top connection, and the template DNA of recycling is carried out with the adapter-primer with sample label
Enrichment Amplification；
S5. the product through step S4 Enrichment Amplifications is recycled；
S6. the sequencing of two generations is carried out to Enrichment Amplification product.
2. sequencing approach according to claim 1, which is characterized in that the fixed distinguished sequence label is located at the 3 ' of P5
End, the reverse complementary sequence of the fixed distinguished sequence label are located at the 5 ' ends of P7.
3. sequencing approach according to claim 1, which is characterized in that the 4-16 kinds contain different fixed distinguished sequence marks
The connector of label is the mixing of equimolecular concentration, wherein, the fixed distinguished sequence label is by 2-10 base composition.
4. sequencing approach according to claim 1, which is characterized in that each sample, which contains specific 4-16 kinds difference, to be fixed
Specially sequence label, specially sequence label is different to the fixation between each sample.
5. sequencing approach according to claim 2, which is characterized in that P5 is the deoxyribonucleoside as shown in SEQ ID NO.1
Acid sequence, the P7 are the deoxynucleotide sequence as shown in SEQ ID NO.2.
6. according to the method described in claim 1, it is characterized in that, the adapter-primer packet of sample label is carried in the step S4
Forward primer and reverse primer are included, the forward primer is the deoxynucleotide sequence as shown in SEQ ID NO.3, described reversed
Primer is the deoxynucleotide sequence as shown in SEQ ID NO.4.
7. a kind of kit realized such as claim 1-6 any one of them sequencing approaches, which is characterized in that including connector,
Connector connection reagent, adapter-primer, the nucleic acid enriching magnetic bead for expanding connection product.
8. kit according to claim 7, which is characterized in that the connector is formed for P5 sequences and P7 sequence anneals,
The P5 is the deoxynucleotide sequence as shown in SEQ ID NO.1, and the P7 is the deoxyribonucleoside as shown in SEQ ID NO.2
Acid sequence.
9. kit according to claim 7, which is characterized in that the adapter-primer for expanding connection product includes forward primer
And reverse primer, the forward primer are the deoxynucleotide sequence as shown in SEQ ID NO.3, the reverse primer is such as
Deoxynucleotide sequence shown in SEQ ID NO.4.
10. the application of the sequencing approach as described in any one of claim 1-6, which is characterized in that the sequencing approach can be single
Detection in Gene Mutation or more applied to microbial diversity sequencing, population is solely either conjointly employed in probe capture technique
Sample is analyzed or the diversity analysis of functional gene.
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| CN111005073A (en) * | 2019-09-29 | 2020-04-14 | 深兰科技（上海）有限公司 | Method and device for constructing multi-sample library |
| CN111118127A (en) * | 2019-12-28 | 2020-05-08 | 郑州大学 | Specific label error-proofing kit for second-generation DNA sequencing sample |
| CN111748613A (en) * | 2019-03-27 | 2020-10-09 | 华大数极生物科技(深圳)有限公司 | Design method and preparation method of double-label joint |
| CN112410331A (en) * | 2020-10-28 | 2021-02-26 | 深圳市睿法生物科技有限公司 | Linker with molecular label and sample label and single-chain library building method thereof |
| CN112592968A (en) * | 2020-12-27 | 2021-04-02 | 苏州科诺医学检验实验室有限公司 | Molecular tag joint for high-throughput sequencing and synthesis method and application thereof |
| CN113444769A (en) * | 2020-03-28 | 2021-09-28 | 深圳人体密码基因科技有限公司 | Construction method and application of DNA tag sequence |
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