CN106350590B

CN106350590B - DNA library construction method for high-throughput sequencing

Info

Publication number: CN106350590B
Application number: CN201610807018.1A
Authority: CN
Inventors: 王旭; 钟嘉泳; 张弓; 董鸣; 余卓
Original assignee: Chaintech Medical (shenzhen) Technology Co Ltd
Current assignee: Chaintech Medical (shenzhen) Technology Co Ltd
Priority date: 2016-09-06
Filing date: 2016-09-06
Publication date: 2019-12-10
Anticipated expiration: 2036-09-06
Also published as: CN106350590A

Abstract

the invention discloses a method for constructing an amplicon sequencing library, which comprises the steps of carrying out multiple PCR on a DNA template obtained from a sample to be tested by using a plurality of pairs of fusion primers, and recovering PCR products to obtain a sequencing library; wherein the multiple pairs of fusion primers are respectively directed to different target fragments on the DNA template, and the fusion primers comprise specific primer sequences and sequencing joint sequences directed to the target fragments; wherein the multiplex PCR is carried out under the following reaction conditions: 2min at 95 ℃; 38 cycles, each cycle is 95 ℃ for 30s, then the temperature is slowly reduced from 76 ℃ to any temperature between 55 ℃ and 58 ℃, the temperature is reduced by 0.1 ℃ per second, the temperature is maintained for 20s after the temperature is reduced to 58 ℃, and then the temperature is 30s at 72 ℃; keeping at 72 deg.C for 2min and 4 deg.C. The invention also discloses application of the library construction method in high-throughput sequencing detection of STR gene loci and paternity test.

Description

DNA library construction method for high-throughput sequencing

Technical Field

The invention relates to the field of biological detection, in particular to a DNA library construction method for high-throughput sequencing.

Background

the main subjects of research and analysis of forensic DNA testing techniques are polymorphisms of DNA in organisms. In general, the location of a DNA marker for a gene or non-coding region on a chromosome is called a locus, and genes with different sequences located at the same locus are called alleles. DNA polymorphism refers to the existence of multiple alleles at a genetic marker locus, and analysis of differences in alleles at a locus is the biological basis for achieving identity [1 ].

short Tandem Repeats (STR) [2] in microsatellite DNA is a DNA polymorphism widely present in 23 pairs of chromosomes of the human genome, and generally consists of repeat units (or core sequences) of 2-6bp, and the difference between different individuals (or alleles) is generally only expressed as the difference in the number of repeats of the repeat units. STR loci are important genetic markers, and are widely used because they have a high level of polymorphism and are easily detected by PCR. STR loci have the following characteristics: the STR has a large number of polymorphic loci, the fragment length is generally less than 400bp, the amplification is easy, and the method is suitable for the detection of trace detection materials; the size of the allele is relatively close, and the preferential amplification of the smaller allele is not obvious; the STR loci of different sites have smaller segment length difference, thereby being convenient for multiplex amplification and improving the detection efficiency.

The STR locus typing detection technology has an important role in forensic DNA detection and paternity identification. Most paternity test laboratories in China use a multi-locus STR typing technology as a main means, analyze and compare different STR locus polymorphisms and calculate an paternity index (PI value, namely the ratio of true father possibility to false father possibility). The standard (PI value is more than or equal to 10000) adopted by most laboratories [3] in the world is used as a boundary line for distinguishing whether the 'existence of relationships' exists.

The traditional STR typing technology is mainly based on a PCR technology and a capillary electrophoresis technology, and multiple PCR amplification is carried out on a sample by using a fluorescence-labeled primer, amplified fragments with different sizes are generated and separated in capillary electrophoresis, so that typing is realized. The Sanger sequencing method is difficult to use for STR typing analysis due to the reasons of throughput and cost.

The second Generation high throughput Sequencing technology (Next Generation Sequencing) can perform Sequencing on hundreds of thousands to millions of DNA molecules at a time, and promotes the rapid development of the Sequencing technology. The whole genome can be sequenced by using a second-generation high-throughput sequencing technology. When a researcher is interested in only a specific genomic region, the Sequencing study can be performed on only the corresponding region using an Amplicon Sequencing (amplification Sequencing) method. The method comprises the steps of designing a primer of a genome region of interest, carrying out PCR amplification, enriching a target region, then carrying out library construction aiming at a PCR product with a specific length or a captured fragment, carrying out high-throughput sequencing and carrying out sequence analysis.

the conventional method for constructing the library is complex in steps, wastes time and large workload, and is not beneficial to sequencing and constructing a library of a large number of samples.

disclosure of Invention

the invention aims to provide a construction method of an amplicon sequencing library and application of the library construction method in high-throughput sequencing detection of STR loci.

According to one aspect of the invention, a method for constructing an amplicon sequencing library is provided, comprising performing multiplex PCR on a DNA template obtained from a sample to be tested by using a plurality of pairs of fusion primers, and recovering PCR products to obtain a sequencing library; wherein the multiple pairs of fusion primers are respectively directed to different target fragments on the DNA template, and each pair of fusion primers sequentially comprises a sequencing joint sequence and a specific primer sequence directed to the target fragment from the 5 'end to the 3' end.

In a preferred embodiment, the multiplex PCR is carried out under the following reaction conditions: 2min at 95 ℃; 38 cycles, each cycle is 95 ℃ for 30s, then the temperature is slowly reduced from 76 ℃ to any temperature between 55 ℃ and 58 ℃, the temperature is reduced by 0.1 ℃ per second, the temperature is maintained for 20s after the temperature is reduced to the target temperature, and then the temperature is reduced for 30s at 72 ℃; 72 ℃ for 2 min.

In the present invention, the "multiple pairs of fusion primers" refers to two, three, or more pairs of fusion primers.

In a more preferred embodiment, the multiplex PCR is carried out under the following reaction conditions: 2min at 95 ℃; 38 cycles, each cycle is 95 ℃ for 30s, then the temperature is slowly reduced from 76 ℃ to 58 ℃ and is reduced by 0.1 ℃ per second, the temperature is reduced to 58 ℃ and then is kept for 20s, and then the temperature is reduced to 72 ℃ for 30 s; 72 ℃ for 2 min.

in some embodiments, the sample to be tested includes, but is not limited to, blood, body fluid, saliva, semen, hair, muscle, or tissue organ. In a preferred embodiment, the DNA template is genomic DNA extracted from a sample to be tested.

In the present invention, each pair of fusion primers comprises a forward primer sequence and a reverse primer sequence specific to the target fragment.

In some embodiments, an index sequence is included in the sequencing adapter sequence to identify different DNA templates, facilitating mixed sequencing of different samples.

the sequencing linker sequence included in the fusion primer of the invention may be: 5'-CCTCTCTATGGGCAGTCGGTGAT-3' in the upstream primer and 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-index sequence-GAT-3' in the downstream primer.

the sequencing linker sequence contained in the fusion primer of the invention may also be: 5 '-AATGATACGGCGACCACCGAGATCTACAC-the first index sequence-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' in the upstream primer, and 5'-CAAGCAGAAGACGGCATACGAGAT + the second index sequence + GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' in the downstream primer.

wherein the first index sequence and the second index sequence are different index sequences.

in some embodiments, the multiplex PCR reaction is performed in 1 or more reaction systems.

In preferred embodiments, two, three, four, or five fusion primer pairs for different fragments of interest are included in a reaction system.

in a preferred embodiment, the fragment of interest is an STR locus.

In a preferred embodiment, the fragments of interest comprise two or more STR loci selected from the following STR loci:

CSF1PO (GenBank X14720), FGA (GenBank M64982), TH01(GenBank D00269), TPOX (GenBank M68651), D3S1358(NT _005997positions 754983-.

in a more preferred embodiment, the fragment of interest includes all of the following STR loci:

Wherein the specific forward primer sequence and reverse primer sequence for each STR locus are as follows:

CSF1 PO: forward direction 5 '-3': TAGCAGGTTGCTAACCACCC, reverse 5 '-3': TCAGACCCTGTTCTAAGTACTTC, respectively;

FGA: forward direction 5 '-3': CCCATAGGTTTTGAACTCACAG, reverse 5 '-3': GTGATTTGTCTGTAATTGCCAGC, respectively;

TH 01: forward direction 5 '-3': GGGCAAAATTCAAAGGGTATCTG, reverse 5 '-3': TGCAGGTCACAGGGAACAC, respectively;

TPOX: forward direction 5 '-3': AGGCACTTAGGGAACCCTC, reverse 5 '-3': TCCTTGTCAGCGTTTATTTGCC, respectively;

D3S 1358: forward direction 5 '-3': CAAGACCCTGTCTCATAGATAG, reverse 5 '-3': TCAACAGAGGCTTGCATGTATC, respectively;

D5S 818: forward direction 5 '-3': GTGACAAGGGTGATTTTCCTCTT, reverse 5 '-3': GTGATTCCAATCATAGCCACAG, respectively;

D7S 820: forward direction 5 '-3': GGTCAGGCTGACTATGGAG, reverse 5 '-3': TCCTCATTGACAGAATTGCACC, respectively;

D8S 1179: forward direction 5 '-3': TCTTTTTGCCCACACGGCC, reverse 5 '-3': CTGTAGATTATTTTCACTGTGGGG, respectively;

D13S 317: forward direction 5 '-3': ATTTCTTTAGTGGGCATCCGTG, reverse 5 '-3': CCTTCAACTTGGGTTGAGCC, respectively;

D16S 539: forward direction 5 '-3': CAGATCCCAAGCTCTTCCTC, reverse 5 '-3': GCATGTATCTATCATCCATCTCTG, respectively;

D18S 51: forward direction 5 '-3': CACTTCACTCTGAGTGACAAATTG, reverse 5 '-3': GTGTGGAGATGTCTTACAATAACAG, respectively;

D21S 11: forward direction 5 '-3': TCAATTCCCCAAGTGAATTGCC, reverse 5 '-3': TGTTCTCCAGAGACAGACTAATAG, respectively;

D2S 1338: forward direction 5 '-3': GTGGATTTGGAAACAGAAATGGC, reverse 5 '-3': GTGGCCCATAATCATGAGTTATTC, respectively;

CD 4: forward direction 5 '-3': AGGGGTACTTGTGTTAATTGTTGG, reverse 5 '-3': GCGTTTTCCAGTCTGAAAAAAGTG, respectively;

D12S 391: forward direction 5 '-3': GAATCAACAGGATCAATGGATGC, reverse 5 '-3': CCTCCATATCACTTGAGCTAATTC, respectively;

FABP: forward direction 5 '-3': TTGTAAGCTCCATGAGGTTAGAG, reverse 5 '-3': AGCCTCCCTAGGTCAGATAG, respectively;

PLA2a 1: forward direction 5 '-3': TAGTATCAGTTTCATAGGGTCACC, reverse 5 '-3': AGTTCGTTTCCATTGTCTGTCC, respectively;

D18S 865: forward direction 5 '-3': CAAATGTAGATCTTGGGACTTGTC, reverse 5 '-3': ATTCTCAAACATCCCCATTACCTTC, respectively;

VWA: forward direction 5 '-3': TCAGTATGTGACTTGGATTGATC, reverse 5 '-3': CAGGTTAGATAGATTAGACAGACAG are provided.

In a preferred embodiment, the multiplex PCR reaction is carried out in 1 or more reaction systems. The plurality of reaction systems includes one or more reaction systems selected from the group consisting of:

a reaction system comprising a fusion primer to the TPOX locus and a fusion primer to the D18S51 locus;

A reaction system comprising a fusion primer to the D8S1179 locus and a fusion primer to the D21S11 locus;

A reaction system comprising a fusion primer for the TPOX locus, a fusion primer for the D18S51 locus, a fusion primer for the D8S1179 locus, and a fusion primer for the D21S11 locus;

A reaction system comprising a fusion primer to the CSF1PO locus, a fusion primer to the D3S1358 locus, a fusion primer to the D13S317 locus, and a fusion primer to the D12S391 locus;

A reaction system comprising a fusion primer for the FGA locus, a fusion primer for the TH01 locus, a fusion primer for the D5S818 locus, and a fusion primer for the PLA2a1 locus;

A reaction system comprising a fusion primer for the FGA locus, a fusion primer for the TH01 locus, a fusion primer for the D5S818 locus, and a fusion primer for the VMA locus;

A reaction system comprising a fusion primer to the D7S820 locus, a fusion primer to the D16S539 locus, a fusion primer to the D2S1338 locus, a fusion primer to the CD4 locus, and a FABP primer; and

A reaction system comprising a fusion primer to the D7S820 locus, a fusion primer to the D16S539 locus, a fusion primer to the D2S1338 locus, a fusion primer to the CD4 locus and a fusion primer to the D18S865 locus.

In a more preferred embodiment, the plurality of reaction systems comprises one or more reaction systems selected from the group consisting of:

a reaction system comprising 1.5 μ M fusion primer to the TPOX locus and 3.5 μ M fusion primer to the D18S51 locus;

a reaction system comprising 1.5 μ M of the fusion primer to the D8S1179 locus and 3.5 μ M of the fusion primer to the D21S11 locus;

A reaction system comprising 1. mu.M fusion primer to the TPOX locus, 1.5. mu.M fusion primer to the D18S51 locus, 1. mu.M fusion primer to the D8S1179 locus, and 1.5. mu.M fusion primer to the D21S11 locus;

A reaction system comprising 1 μ M fusion primer to the CSF1PO locus, 2 μ M fusion primer to the D3S1358 locus, 1 μ M fusion primer to the D13S317 locus, and 1 μ M fusion primer to the D12S391 locus;

A reaction system comprising 1 μ M fusion primer to the CSF1PO locus, 1 μ M fusion primer to the D3S1358 locus, 1 μ M fusion primer to the D13S317 locus, and 2 μ M fusion primer to the D12S391 locus;

a reaction system comprising 1 μ M fusion primer to the FGA locus, 1.5 μ M fusion primer to the TH01 locus, 1.5 μ M fusion primer to the D5S818 locus, and 1 μ M fusion primer to the PLA2A1 locus;

A reaction system comprising 1.5 μ M fusion primer to the FGA locus, 1 μ M fusion primer to the TH01 locus, 1.5 μ M fusion primer to the D5S818 locus, and 1 μ M fusion primer to the VMA locus;

A reaction system comprising 1. mu.M fusion primer to the D7S820 locus, 1. mu.M fusion primer to the D16S539 locus, 1. mu.M fusion primer to the D2S1338 locus, 1. mu.M fusion primer to the CD4 locus, and 1. mu.M FABP primer; and

Reaction system comprising 1. mu.M fusion primer to the D7S820 locus, 1. mu.M fusion primer to the D16S539 locus, 1. mu.M fusion primer to the D2S1338 locus, 1. mu.M fusion primer to the CD4 locus and 1. mu.M fusion primer to the D18S865 locus.

Other STR loci can also be selected as appropriate. Alternative STR loci are selected and are shown in website: http:// www.cstl.nist.gov/biotch/strbase/seq _ info. htm, which is an authoritative website for forensic science that provides STR loci involved in forensic genetic identification. Reference sequence information for these STR loci is available from the website http:// www.cstl.nist.gov/biotech/strbase/seq _ ref.

The method for constructing the amplicon sequencing library is suitable for high-throughput sequencing of any amplicon, for example, the method can be used for detecting STR loci, SNP, SNV, a certain range of insertion deletion mutations and the like through high-throughput sequencing, but is not limited to the above.

Another aspect of the invention provides a method of high throughput sequencing of amplicons, comprising: and preparing a sequencing library by using the amplicon sequencing library construction method, then carrying out high-throughput sequencing and carrying out data analysis on sequencing data.

Target fragments for high throughput sequencing of amplicons include, but are not limited to, fragments comprising STR loci or comprising SNP sites. In a preferred embodiment, the target fragments for amplicon high-throughput sequencing are multiple STR loci.

Another aspect of the present invention provides a method for detecting an STR locus, comprising: the amplicon sequencing library construction method is utilized to prepare a sequencing library aiming at a plurality of STR loci, and then high-throughput sequencing is carried out and data analysis is carried out on sequencing data.

In a preferred embodiment, the data analysis comprises obtaining the number of repeats of the core repeat sequence of the plurality of STR loci.

In some embodiments, the data analysis further comprises analyzing the shadow bands resulting from strand slip mismatches.

in some embodiments, the data analysis further comprises obtaining mutation information in the plurality of STR loci.

In some embodiments, the methods of high throughput sequencing of amplicons and methods of detecting STR loci of the present invention are of non-diagnostic interest.

The method for detecting the STR locus can be used for forensic medicine and other related fields related to the STR locus detection, such as the fields of gene diagnosis, genetic map construction, population genetics research and the like. In a preferred embodiment, the methods of the present invention for detecting STR loci are used for paternity testing.

the invention also provides an paternity test method, which comprises the following steps: the number of repeats in the genome is detected by the detection method of the STR locus described above, and then the paternity index (Pi value) is calculated.

The invention has the beneficial effects that:

(1) The whole library building process can be completed by only one-time multiplex PCR, so that the time and the cost for sequencing and building the library of the amplicon are greatly reduced. When the method is applied to STR locus analysis and paternity relationship identification, the process can be simplified and the cost can be reduced.

(2) The amplicon database construction method of the invention optimizes the multiplex PCR reaction conditions, is suitable for high-throughput sequencing of various amplicons, has no selectivity to primers, and can be used for once amplification database construction by using different primers.

Drawings

FIG. 1 is an agarose gel electrophoresis of the products of the multiplex PCR reaction of example 1.

FIG. 2 is a library quality control chart in example 1.

FIG. 3 is a plot of the distribution of different numbers of repeats in the sequencing data for each STR in example 1.

FIG. 4 is a nucleic acid electrophoresis image of a polyacrylamide PAGE gel of the products of the multiplex PCR reaction in example 2.

FIG. 5 is a library quality control chart in example 2.

Detailed Description

In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various chemicals and biologicals used in the examples were all commercially available products.

example 1: forensic genetic relationship identification

this example aims to detect the number of repeats of a repeated sequence in an STR, using an NGS sequencing protocol.

First, oral epithelial cell collection and treatment

(1) Human oral epithelial cells were collected using an oral swab collection tube.

(2) Oral epithelial cell DNA was extracted using an oral swab genome extraction kit (DP 322).

(3) OD260nm and OD280nm were measured using a Nanodrop 2000 type spectrophotometer, and the purity was confirmed to be high, and the concentration was measured.

Second, design of multiple PCR reaction primer

(1) The primer is designed into a fusion primer, and comprises a specific primer sequence of the target segment and a sequencing joint sequence, wherein the sequence contains an index sequence.

(2) The fusion primer structure is as follows:

An upstream primer: 5'-CCTCTCTATGGGCAGTCGGTGAT-3' + target fragment-specific forward primer;

downstream primer 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG CTAAGGTAAC GAT-3' + target fragment specific reverse primer;

Wherein the underlined sequence is an index sequence.

(3) The STR loci to be amplified and sequenced and the corresponding specific primers are shown in table 1.

TABLE 1STR loci corresponding specific primers

STR loci	Forward primer 5'-3'	reverse primer of 5'-3'
			CSF1PO	TAGCAGGTTGCTAACCACCC	TCAGACCCTGTTCTAAGTACTTC
FGA	CCCATAGGTTTTGAACTCACAG	GTGATTTGTCTGTAATTGCCAGC
			TH01	GGGCAAAATTCAAAGGGTATCTG	TGCAGGTCACAGGGAACAC
TPOX	AGGCACTTAGGGAACCCTC	TCCTTGTCAGCGTTTATTTGCC
			D3S1358	CAAGACCCTGTCTCATAGATAG	TCAACAGAGGCTTGCATGTATC
D5S818	GTGACAAGGGTGATTTTCCTCTT	GTGATTCCAATCATAGCCACAG
			D7S820	GGTCAGGCTGACTATGGAG	TCCTCATTGACAGAATTGCACC
D8S1179	TCTTTTTGCCCACACGGCC	CTGTAGATTATTTTCACTGTGGGG
			D13S317	ATTTCTTTAGTGGGCATCCGTG	CCTTCAACTTGGGTTGAGCC
D16S539	CAGATCCCAAGCTCTTCCTC	GCATGTATCTATCATCCATCTCTG
			D18S51	CACTTCACTCTGAGTGACAAATTG	GTGTGGAGATGTCTTACAATAACAG
D21S11	TCAATTCCCCAAGTGAATTGCC	TGTTCTCCAGAGACAGACTAATAG
			D2S1338	GTGGATTTGGAAACAGAAATGGC	GTGGCCCATAATCATGAGTTATTC
CD4	AGGGGTACTTGTGTTAATTGTTGG	GCGTTTTCCAGTCTGAAAAAAGTG
			D12S391	GAATCAACAGGATCAATGGATGC	CCTCCATATCACTTGAGCTAATTC
FABP	TTGTAAGCTCCATGAGGTTAGAG	AGCCTCCCTAGGTCAGATAG
			PLA2A1	TAGTATCAGTTTCATAGGGTCACC	AGTTCGTTTCCATTGTCTGTCC

Third, multiple PCR reaction library construction and nucleic acid agarose gel electrophoresis

The multiplex PCR reaction was carried out in five groups, and the grouping and the concentration of each primer are shown in Table 2.

TABLE 2 multiplex PCR primer grouping

the reagent used in multiplex PCR was DreamTaq Green PCR Master Mix 2X (Thermo Fisher, cat # K1081), and the reaction system is shown in Table 3:

TABLE 3 multiplex PCR reaction System

The multiple PCR reaction was performed by slow cooling method, and the reaction conditions are shown in Table 4.

TABLE 4 multiplex PCR reaction conditions

And (3) carrying out agarose gel electrophoresis on the PCR reaction product, wherein the conditions of the electrophoresis are as follows: the voltage is 120V, the current is 400mA, the time is 30min, the glue concentration is 1.5%, and the marker strip standard sequentially comprises the following components from bottom to top: 100bp, 200bp, 300bp, 400bp, 500bp, 700bp, 1kb, 1600bp, 2kb, 5kb, 8kb and 10kb, and the results are shown in FIG. 1, 5 groups of multiplex PCR reactions can amplify target bands, and the positions of the target bands are in the expected range.

The premixed five sets of multiplex PCR products were purified using the SanPrep column PCR product purification kit (Shanghai Biotech, cat. No. B518141) and purified according to the manufacturer's instructions to obtain a DNA library for sequencing. The Nanodrop spectrophotometer determined that the purified DNA library had a concentration of 128.73 ng/. mu.l, and a 260/280 value of 1.944.

fourth, library quality inspection

The quality detection of the library is completed by Agilent 2100, the quality detection result is shown in figure 2, the graph shows that the multiplex PCR amplification effect is uniform, the preference of great amplification difference is avoided, and the high-throughput sequencing can be directly carried out.

fifth, Large Scale sequencing

(1) And performing high-throughput sequencing on the prepared DNA sequencing library by using an Ion torrent platform SE400, outputting a result as a FASTQ format file after the sequencing is finished, measuring the sequencing data volume to 67M, and measuring the number of reads matched with the STR locus to be 490536.

Sixthly, processing sequencing data

(1) and (3) intercepting the front and back 20-30 bases of each read to locate the STR locus to which each read belongs, and discarding the read if the read cannot locate the target STR locus. The number of reads matched for each STR is shown in table 5.

TABLE 5 number of reads mapped to each STR locus

(2) As the primer strand or the template strand slips during the primer extension process in the multiplex PCR reaction, a base unpaired loop formed by one repeating unit is caused, namely, the strand slips through a mismatch mechanism, and finally a shadow band (Stutter) is generated. In order to facilitate analysis of influence of shadow bands, the type of the number of times of repetition of the core repeating unit obtained by analysis on reads corresponding to each STR in the sequencing data is finally used as an abscissa, and the percentage of the number of reads corresponding to each type is used as an ordinate to make a histogram, as shown in fig. 3 (a-q).

(4) the number of times each STR locus repeat unit in the test sample is shown in table 6:

TABLE 6 number of repeat units per STR locus

STR loci	Number of repetitions
		CSF1PO	10,11
FGA	21,23
		TH01	7,9
TPOX	8
		D3S1358	16
D5S818	11
		D7S820	10
D8S1179	13,14
		D13S317	8,10
D16S539	9,10
		D18S51	18
D21S11	31,32
		D2S1338	19,23
CD4	5
		D12S391	18
FABP	10
		PLA2A1	11,14

Example 2: detecting to which cell line the unknown cell line belongs

This example uses 2 unknown cell lines and confirms to which cell line the unknown cell line belongs by detecting the number of repeats of the repeat sequence in the STR. Wherein NGS sequencing protocol is used.

Extraction of human cell line DNA

(1) human cell line DNA was extracted using the peripheral blood DNA kit (Qiagen, cat # 937236).

(2) OD260nm and OD280nm were measured by a Nanodrop 2000 type spectrophotometer, and the purity was confirmed to be high, and the final concentration was measured to be 51 ng/. mu.l.

second, multiple PCR reaction library establishment and polyacrylamide PAGE gel nucleic acid electrophoresis

(2) The fusion primer structure is as follows:

Sample one:

an upstream primer: 5'-AATGATACGGCGACCACCGAGATCTACAC-3' + index sequence 1(CTCTCTAT) + ACACTCTTTCCCTACACGACGCTCTTCCGATCT + target fragment-specific forward primer;

A downstream primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3' + index sequence 2(TCGCCTTA) + GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT + fragment-specific reverse primer of interest;

Sample two:

An upstream primer: 5'-AATGATACGGCGACCACCGAGATCTACAC-3' + index sequence 1(TATCCTCT) + ACACTCTTTCCCTACACGACGCTCTTCCGATCT + target fragment specific forward primer;

A downstream primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3' + index sequence 2(CTAGTACG) + GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT + target fragment-specific reverse primer;

(3) The STR loci to be amplified and sequenced and the corresponding specific primers are shown in table 7.

TABLE 7STR loci corresponding specific primers

third, multiple PCR reaction library establishment and polyacrylamide PAGE gel nucleic acid electrophoresis

(1) multiplex PCR reactions were carried out using DreamTaq Green PCR Master Mix (2X) reagent (Saimer Feishale, cat # K1081), and the DNA library construction of the target STR was carried out in 4-panel multiplex PCR reactions, the panels and the respective primer concentrations being shown in Table 8, under the same conditions as in example 1.

TABLE 8 multiplex PCR primer grouping

The multiplex PCR reaction system and reaction conditions were the same as in example 1.

(2) Mixing the products of multiple PCR reactions of two samples together, and performing polyacrylamide PAGE gel nucleic acid electrophoresis, wherein the gel concentration is 8%, the gel running time is 4 hours, the current is 16mA, and the time is 20 minutes; 30mA, 3 hours and 40 minutes. As shown in FIG. 4, the target band was amplified in all of the 4 sets of multiplex PCR reactions, and the positions of the target bands were within the expected range.

Fourthly, purifying PCR products (STR related DNA libraries)

The PCR product mixture in which the products of the various PCR reactions were mixed together for both samples was purified according to the instructions of the SanPrep column PCR product purification kit (Shanghai Biotech, cat. No. B518141) instructions, and the concentration of the purified DNA library was 96.44 ng/. mu.l as measured by a Nanodrop spectrophotometer, and the value of 260/280 was 1.826.

Fifth, quality inspection of library

(1) The quality detection of the library is completed by Agilent 2100, the quality detection result is shown in figure 5, the graph shows that the multiplex PCR amplification effect is uniform, the preference of great amplification difference is avoided, and the high-throughput sequencing can be directly carried out.

sixth, Large Scale sequencing

(1) and (3) carrying out high-throughput sequencing on the purified target DNA library by using an Illumina Miseq platform PE250, and outputting a result as a FASTQ format file after the sequencing is finished, wherein the sequencing data volume is 63.6M.

(2) The number of reads that were measured to match the STR locus was: 686347.

Seventh, sequencing data analysis

(1) Selecting a read with one end capable of covering the STR core repetitive sequence in double-end sequencing as an analysis target, discarding the read at the other end, intercepting the front and back 20-30 bases of each target read so as to locate the STR locus to which each read belongs, and discarding the read if the read cannot be located on the target STR locus. The number of reads matched for each STR is shown in table 9:

TABLE 9 number of reads mapped to each STR locus

STR loci	Number of reads located
		CSF1PO	9646
FGA	58728
		TH01	55099
TPOX	39217
		D3S1358	15413
D5S818	62128
		D7S820	108465
D8S1179	57787
		D13S317	76639
D16S539	45218
		D18S51	13047
D21S11	28294
		D2S1338	15417
CD4	22561
		D12S391	36707
D18S865	12472
		VWA	29509

(2) Regarding the effect of Stutter bands generated during PCR reaction, the number of times of repeat of core repeat unit of STR allele of cell line locus was analyzed according to the method in example 1, and finally analyzed and compared with the STR allele typing results (analyzed target STR: CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, VWA) corresponding to cell lines in ATCC cell bank, and the results are shown in tables 10 and 11.

TABLE 10 number of repeat units per STR locus for an unknown cell line

STR loci	Number of repetitions
		CSF1PO	10
D13S317	11,12
		D16S539	9,13
D5S818	13
		D7S820	10
TH01	8
		TPOX	8,11
VWA	16,17

TABLE 11 number of repeat units per STR locus for another unknown cell line

Therefore, the results of allele typing corresponding to the STR locus can be used to determine that the two unknown cell lines belong to the NCI-H292 cell line and the A549 cell line respectively.

Claims

1. A method for constructing an amplicon sequencing library comprises the steps of carrying out multiple PCR on a DNA template obtained from a sample to be tested by using a plurality of pairs of fusion primers, and recovering PCR products to obtain a sequencing library;

wherein the multiple pairs of fusion primers respectively aim at different target fragments on the DNA template, and each pair of fusion primers sequentially comprises a sequencing joint sequence and a specific primer sequence aiming at the target fragment from the 5 'end to the 3' end;

Wherein the multiplex PCR is carried out under the following reaction conditions: 2min at 95 ℃; 38 cycles, each cycle is 95 ℃ for 30s, then the temperature is slowly reduced from 76 ℃ to any temperature between 55 ℃ and 58 ℃, the temperature is reduced by 0.1 ℃ per second, the temperature is maintained for 20s after the temperature is reduced to the target temperature, and then the temperature is reduced for 30s at 72 ℃; 2min at 72 ℃;

Wherein, the sequence of the sequencing joint contained in the fusion primer is as follows: 5'-CCTCTCTATGGGCAGTCGGTGAT-3' in the upstream primer and 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-index sequence-GAT-3' in the downstream primer; or

The sequence of the sequencing joint contained in the fusion primer is as follows: 5 '-AATGATACGGCGACCACCGAGATCTACAC-the first index sequence-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' in the upstream primer, and 5'-CAAGCAGAAGACGGCATACGAGAT + the second index sequence + GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' in the downstream primer;

wherein the first index sequence and the second index sequence are different index sequences;

wherein, the index sequence is used for identifying different DNA templates, so that different samples can be conveniently subjected to mixed sequencing.

2. The method of claim 1, wherein the DNA template is genomic DNA extracted from a sample to be tested.

3. the method of claim 1, wherein the multiplex PCR reaction is performed in 1 or more reaction systems.

4. a method for high throughput sequencing of amplicons of non-diagnostic interest, comprising: a sequencing library prepared by the method of claim 1 or 2 followed by high throughput sequencing.

5. The sequencing method of claim 4, wherein the target fragments for amplicon high throughput sequencing comprise the following STR loci:

CSF1PO, FGA, TH01, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D2S1338, CD4, D12S391, PLA2A1 and FABP.

6. a method for detecting an STR locus of non-diagnostic interest, comprising: preparing a sequencing library for a plurality of STR loci using the amplicon sequencing library construction method of any one of claims 1 to 3, followed by high throughput sequencing and data analysis of the sequencing data.

7. The detection method of claim 6, wherein the data analysis comprises obtaining a number of repetitions of the plurality of STR locus core repeat sequences.

8. The detection method of claim 7, wherein the data analysis further comprises analyzing shadow bands resulting from strand slip mismatches.

9. a method of paternity testing comprising: determining the number of repeats of an STR locus in a genome using the method of claim 7 or 8, and then calculating an paternity index (Pi value).