CN109593758B - Multiplex primer set and method for constructing human B cell immune repertoire based on high-throughput sequencing by using same - Google Patents

Multiplex primer set and method for constructing human B cell immune repertoire based on high-throughput sequencing by using same Download PDF

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CN109593758B
CN109593758B CN201811597255.5A CN201811597255A CN109593758B CN 109593758 B CN109593758 B CN 109593758B CN 201811597255 A CN201811597255 A CN 201811597255A CN 109593758 B CN109593758 B CN 109593758B
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李艳艳
邱盟轩
靖相密
汪朝晖
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Shandong Acv Biotech Co ltd
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Abstract

The invention discloses a multiple primer group and a method for constructing a human B cell immune repertoire by using the primer group based on high-throughput sequencing, belonging to the field of molecular biology detection. The multiplex primer group consists of a group of upstream primers and a group of downstream primers, wherein the upstream primers are formed by connecting a joint 1, a primer bar code 1, a sequencing primer 1 and a specific primer V1-V18 designed aiming at a variable region V region in series; the downstream primer is formed by connecting a joint 2, a primer bar code 2, a sequencing primer 2 and specific primers Va, Vd, Ve, Vg and Vm designed aiming at conserved sequences of C regions of gene constant regions of IgA, lgD, IgE, IgG and IgM in series; the primer bar code 1 and the primer bar code 2 have different sequences; the sequencing primer 1 has the same sequence as the sequencing primer 2. The method can be used for constructing the BCR immune repertoire based on the DNA sample or the RNA sample, and can comprehensively cover the diversity information of the BCR. And the construction efficiency is high and the cost is low.

Description

Multiplex primer set and method for constructing human B cell immune repertoire based on high-throughput sequencing by using same
Technical Field
The invention relates to the field of molecular biology detection, in particular to a multiple primer set and a method for constructing a human B cell immune repertoire based on high-throughput sequencing by using the primer set.
Background
The immune repertoire is the sum of all functionally diverse B and T cells in the circulatory system of an individual at any given time.
Human lymphocytes mainly include T cells and B cells. The B cell antigen receptor (BCR) is a membrane surface immunoglobulin (SmIg) for B cell to recognize antigen and has antigen binding specificityOnly V-region and C-region. The V region consists of two domains, VH and VL, each of which consists of three complementarity determining regions (CDR1, CDR2 and CDR3), the amino acid composition and the arrangement order of the CDRs are highly diverse and can be as high as 10 in one and the same body9～1012The BCR library with huge capacity is formed, individuals are endowed with huge potentials for recognizing various antigens and generating specific antibodies, and the three CDRs are all involved in the recognition of the antigens and jointly determine the antigen specificity of the BCR. It is this diversity that plays a vital role in health, and the more subtypes of immune proteins, the more effective against pathogens, the fewer subtypes the more susceptible to disease. In addition, many other factors, such as age, environment, disease induction and medication, affect the diversity of the immune repertoire.
The construction methods of immune repertoires such as SSCP technology, GeneScan technology, fluorescence quantitative dissolution curve technology and the like have narrow coverage range, can not comprehensively and uniformly reflect the diversity of the immune repertoire, and the high-throughput sequencing can meet the requirements of huge and diverse sequence sequencing.
In the experimental procedure of high throughput sequencing, the preparation of a sequencing library is a very critical step. Due to the diversity of BCR, the amplification preference of primers, the difference of abundance of templates and other factors, the primer design and PCR amplification method are particularly important when building libraries. In addition, the preparation of the traditional sequencing library requires the procedures of sample nucleic acid extraction, enzyme pretreatment or mechanical shearing, joint addition for connection, PCR amplification and the like, and the operation is complex.
Therefore, the BCR immune repertoire construction method based on high-throughput sequencing is provided, so that the experimental operation steps are simplified, the amplification preference of the PCR primers is reduced, and the amplification efficiency is improved.
Disclosure of Invention
In order to make up for the deficiencies of the prior art, the present invention provides a method for constructing a human B cell immune repertoire based on high-throughput sequencing.
The technical scheme of the invention is as follows:
a multiplex primer group consists of a group of upstream primers and a group of downstream primers, wherein the upstream primers are formed by connecting a joint 1, a primer bar code 1, a sequencing primer 1 and a specific primer V1-V18 designed aiming at a variable region V region in series; the downstream primer is formed by connecting a joint 2, a primer bar code 2, a sequencing primer 2 and specific primers Va, Vd, Ve, Vg and Vm designed aiming at conserved sequences of C regions of gene constant regions of IgA, lgD, IgE, IgG and IgM in series; the sequences of the primer bar code 1 and the primer bar code 2 are different; the sequencing primer 1 has the same sequence as the sequencing primer 2.
Preferably, linker 1 has sequence AAT GAT ACG GCG ACC ACC GAG ATC TAC AC; linker 2 sequence CAA GCA GAA GAC GGC ATA CGA GAT.
Preferably, the sequence of the sequencing primer 1 and the sequence of the sequencing primer 2 are GCT CTT CCG ATC T.
As a preferred scheme, the primer bar code 1 and the primer bar code 2 consist of 6 or 8 nucleotides, and at least one nucleotide difference exists between the primer bar code 1 and the primer bar code 2 of different samples.
The primer bar code 1 and the primer bar code 2 can be TACGTA, TGAGCG, TACAGT, CATGTC, etc., or TCCGTTCG, TGTGCGCT, TACCCTCA or GATTCATC.
Preferably, the specific primers V1-V18 in the upstream primer are respectively:
V1：5′-GCTGGGTGCGCCAGATGCCC
V2：5′-TGGATCCGTCAGCCCCCAGG
V3：5′-TGGATCCGTCAGCCCCCGGG
V4：5′-GTGCGACAGGCCCCTGGACAA
V5：5′-GTYCCGCAGGCCCCCGSACAA
V6：5′-GGGTGMGACTCGCTCGRGGACAA
V7：5′-GTGGCACAYGCCCCCGGACAA
V8：5′-GTGCGACAGGCWCGGAGACAA
V9：5′-TCCGCCAGCKCGGAGGGAAGG
V10：5′-TCCGRCTGCCCGCYCCGAA
V11：5′-TCCGGCAGMCGCCCCGGAA
V12：5′-TCCGGCAGCCCGCTCYGAAGG
V13：5′-GMAGCGCCAGSCTCCAGGGAA
V14：5′-GGAGKGCCAGGCTTCSGGGAA
V15：5′-GAAGCGCCWGGCTCCAGGGAA
V16：5′-GGAGCGCCAGGCTCCAMGGAA
V17：5′-GGAGCGCRAGGCTCCGGGCAA
V18：5′-GGAGCGCCAGCCTCYAGGGAA；
specific primers Va, Vd, Ve, Vg and Vm in the downstream primer are respectively as follows:
Vg：5-ATGAYCGATGGGCCCTTGT
Va：5-GCAGACCTTRGGGCTGGTCA
Vm：5-GCGAATTCTCACWGGAGACG
Vd：5-GCGTGSCTGCAGCCTGATA
Ve：5-GTAGAYGGATGGGCTCSGT。
the method for constructing the human B cell immune repertoire based on high-throughput sequencing by utilizing the multiple primer group comprises the following steps:
1) extracting sample nucleic acid DNA or RNA;
2) carrying out first round PCR amplification on sample nucleic acid DNA or RNA to obtain a PCR amplification product 1;
3) recovering a PCR amplification product 1;
4) taking the recovered product of the PCR amplification product 1 as a template to carry out second round of PCR amplification to obtain a PCR amplification product 2;
5) recovering the PCR amplification product 2, wherein the recovered product forms a BCR sequencing library;
6) library detection:
the purity and the size of the obtained BCR sequencing library are detected by an Aglilent 2100 Bioanalyser; determining the concentration of the BCR library by using Nanodrop one;
7) high-throughput sequencing:
sequencing the purified BCR library by using Hiseq2500 of Illumina company, wherein the sequencing mode is PE 150;
8) and (3) data analysis:
after the sequencing data are filtered to remove the sequencing background, V \ D \ J gene comparison analysis is carried out on the off-line data and IMGT immune cell receptor database data, and a B cell immune repertoire spectrum is established.
Preferably, when the sample is DNA, the first round is performed in step 2)The PCR amplification system for PCR amplification is prepared as follows: 5 XPCR Buffer 5.0 μ L, dNTP Mix final concentration 0.5-1mM, multiplex PCR primer set upstream primer set 3-5 μ M, downstream primer set 3-5 μ M, Mg2+Final concentration 3-4mM, Taq enzyme 1.5-3unit, DNA sample 200-.
Further, the amplification procedure of the first round of PCR amplification is as follows:
activating the hot start enzyme at 95 ℃ for 10-20 min; denaturation at 94-95 deg.C for 15sec, annealing at 55-60 deg.C for 60-120sec, and extension at 72 deg.C for 30-40sec, and performing 6-10 cycles; denaturation at 94-95 deg.C for 15sec, annealing at 65-70 deg.C for 40-60sec, and extension at 72 deg.C for 30-40sec, and performing 10-20 cycles; storing at 16 ℃.
Preferably, when the sample is RNA, in step 2), the PCR amplification system for the first round of PCR amplification is prepared as follows: 5 xRT-PCR Buffer 5.0 uL, dNTP Mix final concentration 0.5-1mM, multiplex PCR primer set upstream primer set 3-5 uM, downstream primer set 3-5 uM, RNase inhibitor 0.25 uL, Mg2+The final concentration is 3-4mM, the reverse transcriptase and Taq enzyme mixture is 2.0-3.5unit, the RNA sample is 200-.
Further, the amplification procedure of the first round of PCR amplification is as follows:
reverse transcription is carried out for 35-40min at the temperature of 45-50 ℃; activating the hot start enzyme at 95 ℃ for 10-20 min; denaturation at 94-95 deg.C for 15sec, annealing at 55-60 deg.C for 60-120sec, and extension at 72 deg.C for 30-40sec, and performing 6-10 cycles; denaturation at 94-95 deg.C for 15sec, annealing at 65-70 deg.C for 40-60sec, and extension at 72 deg.C for 30-40sec, and performing 10-20 cycles; storing at 16 ℃.
Preferably, in the steps 3) and 5), the recovery of the PCR amplification product 1 and the recovery of the PCR amplification product 2 are purified and recovered by agarose gel electrophoresis, purification column purification or magnetic bead purification.
Preferably, in step 4), the PCR amplification system for the second round of PCR amplification is prepared as follows:
5 XPCR Buffer 5.0 uL, dNTP Mix final concentration 0.2-0.4mM, linker 1 final concentration 0.2-0.5 uM, linker 2 final concentration 0.1-0.2 uM, Mg2+Final concentration 2-2.5mM, Taq enzyme 1.5-2unit, PCR amplification product 1 recovery product 5 uL, addDEPC water was added to make up the volume to 50. mu.L.
Further, the amplification procedure of the second round of PCR amplification is as follows:
activating the hot start enzyme at 95 ℃ for 10-20 min; denaturation at 94-95 ℃ for 10-20 sec, annealing at 50-55 ℃ for 30-40sec, extension at 72 ℃ for 30-40s, performing 30-40 cycles, and storing at 16 ℃.
Preferably, the upstream primer group of the multiplex PCR primer group is formed by mixing the following 18 upstream primers:
1：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGCTGGGTGCGCCAGATGCCC
2：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTGGATCCGTCAGCCCCCAGG
3：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTGGATCCGTCAGCCCCCGGG
4：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTGCGACAGGCCCCTGGACAA
5：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTYCCGCAGGCCCCCGSACAA
6：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGGTGMGACTCGCTCGRGGACAA
7：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTGGCACAYGCCCCCGGACAA
8：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTGCGACAGGCWCGGAGACAA
9：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGCCAGCKCGGAGGGAAGG
10：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGRCTGCCCGCYCCGAA
11：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGGCAGMCGCCCCGGAA
12：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGGCAGCCCGCTCYGAAGG
13：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGMAGCGCCAGSCTCCAGGGAA
14：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGKGCCAGGCTTCSGGGAA
15：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGAAGCGCCWGGCTCCAGGGAA
16：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGCGCCAGGCTCCAMGGAA
17：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGCGCRAGGCTCCGGGCAA
18：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGCGCCAGCCTCYAGGGAA；
wherein NNNN (NN) in the upstream primer group represents primer barcode 1;
the downstream primer group is formed by mixing the following 5 downstream primers:
1：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN)GCT CTT CCG ATC TATGAYCGATGGGCCCTTGT
2：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN)GCT CTT CCG ATC TGCAGACCTTRGGGCTGGTCA
3：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN)GCT CTT CCG ATC TGCGAATTCTCACWGGAGACG
4：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN)GCT CTT CCG ATC TGCGTGSCTGCAGCCTGATA
5：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN)GCT CTT CCG ATC TGTAGAYGGATGGGCTCSGT；
wherein NNNN (NN) in the downstream primer represents primer barcode 2.
Further, the upstream primer group is formed by mixing 18 upstream primers in an equimolar way; the downstream primer group is formed by mixing 5 downstream primers in an equimolar way.
Preferably, the total number of moles of the forward primer set is the same as the total number of moles of the backward primer set.
The invention has the beneficial effects that:
the method can be used for constructing the BCR immune repertoire based on the DNA sample or the RNA sample, millions of BCR sequences can be obtained after high-throughput sequencing, and the diversity information of the BCR can be comprehensively covered. In terms of the operation method, the primer bar codes and the sequencing primers are directly introduced into the constructed library in a PCR amplification mode, so that the complexity of library construction steps is reduced, the construction efficiency is improved, the time is saved, and the cost consumption of labor, reagents and the like is reduced. The PCR amplification through the two-step method also reduces the skewness of the multiplex PCR amplification and better reflects the real state of the BCR diversity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic diagram of the construction of a human B cell immune repertoire based on high throughput sequencing according to the present invention;
FIG. 2 shows the purity and size of the BCR library obtained in example 2 measured by an Aglilent 2100 Bioanalyser;
FIG. 3 is a CDR3 length distribution diagram in example 2; wherein the horizontal axis represents the nucleotide length of the CDR3, and the vertical axis represents the proportion of the CDR3 with the corresponding length;
FIG. 4 is a diagram of the sequence analysis of the high abundance CDR3 in example 2;
FIG. 5 shows the purity and size of the BCR library obtained in example 3 measured by an Aglilent 2100 Bioanalyser;
FIG. 6 is a CDR3 length distribution diagram in example 3; wherein the horizontal axis represents the nucleotide length of the CDR3, and the vertical axis represents the proportion of the CDR3 with the corresponding length;
FIG. 7 is the sequence analysis chart of the high abundance CDR3 in example 3.
Detailed Description
Description of materials and reagents
Healthy volunteer subjects gave informed consent. Specifically, the reagents adopted in the invention are all commercially available products, and the databases adopted in the embodiment of the invention are all public online databases.
Example 1
A multiplex primer group consists of a group of upstream primers and a group of downstream primers, wherein the upstream primers are formed by connecting a joint 1, a primer bar code 1, a sequencing primer 1 and a specific primer V1-V18 designed aiming at a variable region V region in series; the downstream primer is formed by connecting a joint 2, a primer bar code 2, a sequencing primer 2 and specific primers Va, Vd, Ve, Vg and Vm designed aiming at conserved sequences of C regions of gene constant regions of IgA, lgD, IgE, IgG and IgM in series. The sequences of the primer bar code 1 and the primer bar code 2 are different;
linker 1 sequence AAT GAT ACG GCG ACC ACC GAG ATC TAC AC;
linker 2 sequence CAA GCA GAA GAC GGC ATA CGA GAT.
The sequences of the sequencing primer 1 and the sequencing primer 2 are both GCT CTT CCG ATC T.
The primer bar code 1 and the primer bar code 2 are represented by NNNN (NN) and consist of 6 or 8 nucleotides, so that the on-machine sequencing of different sample libraries is facilitated, the primer bar code sequences of different samples have at least one nucleotide difference, and the primer bar code 1 and the primer bar code 2 have different sequences.
The primer bar code 1 and the primer bar code 2 are TACGTA, TGAGCG, TACAGT, CATGTC and the like or TCCGTTCG, TGTGCGCT, TACCCTCA or GATTCATC.
Specific primers were designed according to the following method: the human BCR CDR3 sequence disclosed in IMGT database is used as a reference sequence, all variable region V region (variable) and gene constant region (constant) C region genes of IgA, lgD, IgE, IgG and IgM are compared and analyzed, Primer Premier 5.0 is used for Primer design, oligo7 is used for Primer dimer and stem-loop mismatch analysis, 18 upstream primers V1-V18 are designed for variable region V region conserved sequence, and 1 downstream Primer Va, Vd, Ve, Vm and Vm are respectively designed for gene constant region (constant) C region gene conserved sequences of IgA, lgD, IgE, IgG and IgM.
TABLE 1 specific primer set
Figure BDA0001921623740000081
Figure BDA0001921623740000091
Note: r is A/G, Y is C/T, M is A/C, K is G/T, S is C/G, W is A/T
Therefore, the sequences of the multiplex primer set used for library construction are shown in Table 2.
TABLE 2 multiplex amplification primer sets
Figure BDA0001921623740000092
Figure BDA0001921623740000101
The invention provides a library construction method of a human B cell immune repertoire, which mainly comprises the use proportion of primers and a PCR amplification program. The sample used in the present invention may be a DNA sample or an RNA sample.
When a DNA sample is used, the sequence of interest is amplified by multiplex PCR for library construction, and two rounds of PCR are performed.
When an RNA sample is used, the library construction can be amplified by using a two-step RT-PCR method, namely, reverse transcription is performed by using reverse transcriptase to synthesize cDNA, and then synthesis of second strand DNA is performed; library construction can also be performed using one-step RT-PCR amplification. The following examples select one-step RT-PCR for multiplex PCR amplification. However, the person skilled in the art can deduce the two-step RT-PCR amplification method from the one-step RT-PCR amplification method without substantial effort.
Example 2
Library construction using DNA samples, sequences of interest were amplified by multiplex PCR:
1. rapid isolation of peripheral blood lymphocytes
1) And (4) checking: taking out lymphocyte anticoagulation separation tube, observing whether there is free separation liquid on separation gel, if there is, 2000g, centrifuging for 1min at room temperature.
2) Sampling: 5ml of peripheral blood was added to the anticoagulation separation tube.
3) Centrifuging: 800g, soft centrifugation for 15min at normal temperature.
4) The plasma layer is aspirated off with a pasteur pipette or pipettor to the point adjacent to the PBMC cell layer, and the PBMCs (buffy coat: between the separation gel and plasma) and transferred to a new 15ml centrifuge tube.
5) Adding physiological saline or 1 × PBS to 15ml, washing 1 time, 300g, and centrifuging 10min at normal temperature soft.
6) Discarding supernatant, adding 2ml of physiological saline or 1 × PBS, re-suspending, adding to 5ml, mixing well and counting.
7)300G, centrifugation for 10min, discarding the supernatant, and adjusting the cell concentration by count to 20 x 106/ml。
2. Sample nucleic acid DNA extraction
The DNA is extracted using a commercial kit, such as QIAGEN blood, tissue DNA extraction kit (CAT: 65904), or genomic DNA by phenol chloroform extraction. In this embodiment, DNA is extracted using QIAGEN blood and tissue DNA extraction kit (CAT: 65904), which is well known to those skilled in the art and will not be described herein.
After the extraction of DNA, the purity and concentration of DNA are determined by using a Nanodrop one spectrophotometer, and the quality of DNA is detected by 1% agarose electrophoresis.
The extracted DNA was determined to have a concentration of 198ng/ul and an OD260/280 of 1.93.
3. First round PCR amplification
The following were added to the PCR tube: 5 XRT-PCR Buffer 5.0. mu.L, dNTP Mix final concentration 0.7mM, multiplex PCR primer set upstream primer set 2.7. mu.M, downstream primer set 2.7. mu.M, Mg2+Final concentration 3mM, Taq enzyme 1.5unit, DNA sample 2.5 μ L, DEPC water was added to make up the volume to 25 μ L.
Amplification was performed according to the following amplification conditions to obtain PCR amplification product 1:
activating the hot start enzyme at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, annealing at 58 ℃ for 120sec, and extension at 72 ℃ for 40sec, for 10 cycles; denaturation at 95 ℃ for 15sec, annealing at 70 ℃ for 60sec, and extension at 72 ℃ for 30sec, for 20 cycles; storing at 16 ℃.
4. Recovery of PCR amplification product 1
And purifying and recovering the PCR amplification product 1 by using a magnetic bead purification mode, wherein the recovered product is used as a template for the second round of PCR amplification. The recovery and purification of the product removes primers, dNTP, enzyme and the like which do not participate in the reaction, and also eliminates the influence of the factors on the second round of PCR amplification.
The magnetic bead purification comprises the following specific steps:
1) taking out the magnetic beads, shaking and mixing uniformly for 5min, taking out 45 mu l of sample, and placing the sample at room temperature for 10 min;
2) the PCR amplification product 1 was removed, and 30. mu. l H was added thereto2O, mixing uniformly, adding 45 mu l of diluted PCR amplification product into the magnetic beads after the room temperature is balanced, sucking and blowing for 10 times by using a gun, mixing uniformly, and placing at the room temperature for 2 min;
3) opening the cover and placing on a magnetic frame for 1min, clarifying the liquid, sucking out the liquid, adding 125 μ l 85% ethanol, washing out after 1min, discarding, and air drying the magnetic beads at room temperature for 8 min;
4) add 30. mu.l dd H2O for redissolution.
5. Second round of PCR amplification
The following were added to the PCR tube: 5 XPCR Buffer 5.0 uL, dNTP Mix final concentration of 0.3 mM, 1 adaptor final concentration of 0.35 uM, 2 adaptor final concentration of 0.1 uM, Mg2+, final concentration of 2mM, Taq enzyme 1.8unit, PCR amplification product 1 recovery product 5 uL, adding DEPC water to make up the volume to 50 uL.
Amplification was performed under the following amplification conditions to obtain PCR amplification product 2.
Activating the hot start enzyme at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, annealing at 55 ℃ for 40sec, and elongation at 72 ℃ for 30sec, and storage at 16 ℃ for 35 cycles.
6. PCR amplification product 2 recovery
The obtained PCR amplification product 2 is purified and recovered by a magnetic bead purification method, and the specific purification method refers to step 4. The recovered product thus obtained constitutes a BCR sequencing library. The recovered product eliminates primers, dNTP and the like which do not react, and can further improve the sequencing efficiency and accuracy of the constructed sequencing library.
7. Library detection
The purity and the size of the obtained BCR library are detected by an Aglilent 2100Bioanalyser, the detection result is shown in figure 2, the size of the library is 355bp, the purity of the library is high, and other non-specific amplification sequences are not shown. The BCR library concentration was determined using Nanodrop one, and the recovered library concentration was 21 ng/ul.
8. High throughput sequencing
The purified BCR library was delivered to sequencing company for sequencing using Hiseq2500 from Illumina, in the sequencing mode of PE 150.
9. Data analysis
And (3) after original data obtained by sequencing is subjected to decontamination, joint removal and low-quality removal filtration, comparing off-line data with a reference sequence on the IMGT, and searching corresponding gene segments to obtain information such as VDJ gene frequency, clone frequency distribution, specific BCR sequence, number and the like and establish a B cell immune repertoire. Part of the data results are shown in table 3.
TABLE 3 library sequencing result count
Figure BDA0001921623740000121
Note:
1) sample: a sample name;
2) total reads: number of all reads (sequences) sequenced from the sample;
3) used reads: the number of reads (sequences) that were in turn cloned intact in the sample;
4) percent (%): the percentage of Used reads to Total reads;
5) clones: number of clones in the sample.
The CDR3 length distribution is shown in fig. 3, where the horizontal axis represents the nucleotide length of CDR3 and the vertical axis represents the proportion of CDR3 of the corresponding length. The sequence analysis of the high abundance CDR3 is shown in fig. 4.
The top 10 CDR3 sequences were searched in the immune repertoire side by side for the most abundant (highest number of reads). Wherein the horizontal axis represents the length of the CDR3, the vertical axis represents the frequency of occurrence of the sequences of the corresponding length, and the 10 sequences with the highest frequency are listed in the legend (decreasing in frequency from top to bottom).
Example 3
When RNA samples are used, the sequence of interest is amplified by multiplex RT-PCR for library construction:
1. rapid separation of peripheral blood lymphocytes
1) And (4) checking: taking out lymphocyte anticoagulation separation tube, observing whether there is free separation liquid on separation gel, if there is, 2000g, centrifuging for 1min at room temperature.
2) Sampling: 5ml of peripheral blood was added to the anticoagulation separation tube.
3) Centrifuging: 800g, soft centrifugation for 15min at normal temperature.
4) The plasma layer is aspirated off with a pasteur pipette or pipettor to the point adjacent to the PBMC cell layer, and the PBMCs (buffy coat: between the separation gel and plasma) and transferred to a new 15ml centrifuge tube.
5) Adding physiological saline or 1 × PBS to 15ml, washing 1 time, 300g, and centrifuging 10min at normal temperature soft.
6) Discarding supernatant, adding 2ml of physiological saline or 1 × PBS, re-suspending, adding to 5ml, mixing well and counting.
7)300G, centrifugation for 10min, discarding the supernatant, and adjusting the cell concentration by count to 20 x 106/ml。
2. Sample nucleic acid RNA extraction
RNA extraction was performed using a commercial Kit, such as QIAGEN RNeasy midi Kit (CAT: 75142), or total RNA extraction using Trizol method. In this embodiment, total RNA is extracted by Trizol method, and the extraction procedure is well known to those skilled in the art, and will not be described herein.
After the extraction of RNA, the concentration of RNA is determined by using a Nanodrop one spectrophotometer, and the quality of RNA is detected by 1% agarose electrophoresis.
The concentration of extracted RNA was 156ng/ul, and OD260/280 was 2.01.
3. First round PCR amplification
The following were added to the PCR tube: 5 XRT-PCR Buffer 5.0 μ L, dNTP Mix final concentration 0.8 mM, multiplex PCR primer set upstream primer set 1.8 μ M, downstream primer set 1.8 μ M, RNase inhibitor 0.25 μ L, Mg2+Final concentration 3.5mM, reverse transcriptase, Taq enzyme mix 2.5unit, RNA sample 3.5 μ L, add DEPC water to make up volume to 25 μ L.
Amplification was performed according to the following amplification conditions to obtain PCR amplification product 1:
reverse transcription is carried out for 35min at 50 ℃; activating the hot start enzyme at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, annealing at 58 ℃ for 120sec, and extension at 72 ℃ for 40sec, for 8 cycles; denaturation at 95 ℃ for 15sec, annealing at 69 ℃ for 60sec, and extension at 72 ℃ for 30sec, for 15 cycles; storing at 16 ℃.
4. Recovery of PCR amplification product 1
And purifying and recovering the PCR amplification product 1 by using a magnetic bead purification mode, wherein the recovered product is used as a template for the second round of PCR amplification. The recovery and purification of the product removes primers, dNTP, enzyme and the like which do not participate in the reaction, and also eliminates the influence of the factors on the second round of PCR amplification.
The magnetic bead purification comprises the following specific steps:
1) taking out the magnetic beads, shaking and mixing uniformly for 5min, taking out 45 mu l of sample, and placing the sample at room temperature for 10 min;
2) the PCR amplification product 1 was removed, and 30. mu. l H was added thereto2O, mixing uniformly, adding 45 mu l of diluted PCR amplification product into the magnetic beads after the room temperature is balanced, sucking and blowing for 10 times by using a gun, mixing uniformly, and placing at the room temperature for 2 min;
3) opening the cover and placing on a magnetic frame for 1min, clarifying the liquid, sucking out the liquid, adding 125 μ l 85% ethanol, washing out after 1min, discarding, and air drying the magnetic beads at room temperature for 8 min;
4) add 30. mu.l dd H2O for redissolution.
5. Second round of PCR amplification
The following were added to the PCR tube: 5 XPCR Buffer 5.0 uL, dNTP Mix final concentration 0.3 mM, thenFinal concentration of 0.35. mu.M for head 1, 0.2. mu.M for linker 2, Mg2+Final concentration 2mM, Taq enzyme 1.8unit, PCR amplification product 1 recovery product 5 μ L, add DEPC water to make up volume to 50 μ L.
Amplification was performed under the following amplification conditions to obtain PCR amplification product 2.
Activating the hot start enzyme at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, annealing at 53 ℃ for 35sec, and elongation at 72 ℃ for 30sec, and storage at 16 ℃ for 35 cycles.
6. PCR amplification product 2 recovery
The obtained PCR amplification product 2 is purified and recovered by a magnetic bead purification method, and the specific purification method refers to step 4. The recovered product thus obtained constitutes a BCR sequencing library. The recovered product eliminates primers, dNTP and the like which do not react, and can further improve the sequencing efficiency and accuracy of the constructed sequencing library.
Note: the PCR amplification product 2 was 50. mu.L, and was recovered in two portions.
7. Library detection
The purity and the size of the obtained BCR library are detected by an Aglilent 2100Bioanalyser, the detection result is shown in figure 5, the size of the library is 356bp, the purity of the library is high, and other non-specific amplification sequences are not shown. The BCR library concentration was determined using Nanodrop one and the recovered library concentration was 19 ng/ul.
8. High throughput sequencing
The purified BCR library was delivered to sequencing company for sequencing using Hiseq2500 from Illumina, in the sequencing mode of PE 150.
9. Data analysis
After the sequencing data are filtered to remove the sequencing background, V \ D \ J gene comparison is carried out on the off-line data and IMGT immune cell receptor database data, and information such as VDJ gene frequency, clone frequency distribution, special BCR sequence, number and the like is obtained by searching corresponding gene segments to establish a T cell immune repertoire. Partial data results are shown in table 4.
TABLE 4 library sequencing result counts
Figure BDA0001921623740000151
Note:
1) sample: a sample name;
2) total reads: number of all reads (sequences) sequenced from the sample;
3) used reads: the number of reads (sequences) that were in turn cloned intact in the sample;
4) percent (%): the percentage of Used reads to Total reads;
5) clones: number of clones in the sample.
The CDR3 length distribution is shown in fig. 6, where the horizontal axis represents the nucleotide length of CDR3 and the vertical axis represents the proportion of CDR3 of the corresponding length. The sequence analysis of the high abundance CDR3 is shown in fig. 7.
The top 10 CDR3 sequences were searched in the immune repertoire side by side for the most abundant (highest number of reads). Wherein the horizontal axis represents the length of the CDR3, the vertical axis represents the frequency of occurrence of the sequences of the corresponding length, and the 10 sequences with the highest frequency are listed in the legend (decreasing in frequency from top to bottom).
The B cell immune repertoire has wide application fields, the diversity of the T cell immune repertoire is measured through the huge flux of a high-throughput sequencing platform, complete diversity information of an individual in a certain specific period can be provided, and the change of an antibody and BCR in the adaptive immune process is researched. The sequencing of the B cell immune repertoire can be applied to the fields of research and development of vaccines and medicines, discovery of biomarkers, detection of minimal residual disease, research of autoimmune diseases, post-transplantation monitoring and the like.
SEQUENCE LISTING
<110> Shandong Aickvir Biotechnology Ltd
<120> multiplex primer set and method for constructing human B cell immune repertoire based on high-throughput sequencing by using same
<130> 2018
<160> 49
<170> PatentIn version 3.5
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aatgatacgg cgaccaccga gatctacac 29
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caagcagaag acggcatacg agat 24
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gctcttccga tct 13
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gctgggtgcg ccagatgccc 20
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tggatccgtc agcccccagg 20
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tggatccgtc agcccccggg 20
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gtgcgacagg cccctggaca a 21
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gtyccgcagg cccccgsaca a 21
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gggtgmgact cgctcgrgga caa 23
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gtggcacayg cccccggaca a 21
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gtgcgacagg cwcggagaca a 21
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tccgccagck cggagggaag g 21
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tccgrctgcc cgcyccgaa 19
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tccggcagmc gccccggaa 19
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tccggcagcc cgctcygaag g 21
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gmagcgccag sctccaggga a 21
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ggagkgccag gcttcsggga a 21
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gaagcgccwg gctccaggga a 21
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ggagcgccag gctccamgga a 21
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ggagcgcrag gctccgggca a 21
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ggagcgccag cctcyaggga a 21
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atgaycgatg ggcccttgt 19
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gcagaccttr gggctggtca 20
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gcgaattctc acwggagacg 20
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gcgtgsctgc agcctgata 19
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gtagayggat gggctcsgt 19
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gctgggtgcg 60
ccagatgccc 70
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tggatccgtc 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tggatccgtc 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtgcgacagg 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtyccgcagg 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gggtgmgact 60
cgctcgrgga caa 73
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtggcacayg 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtgcgacagg 60
cwcggagaca a 71
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccgccagck 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccgrctgcc 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccggcagmc 60
gccccggaa 69
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccggcagcc 60
cgctcygaag g 71
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gmagcgccag 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagkgccag 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gaagcgccwg 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagcgccag 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagcgcrag 60
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagcgccag 60
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caagcagaag acggcatacg agatnnnnnn nngctcttcc gatctgcgtg sctgcagcct 60
gata 64
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caagcagaag acggcatacg agatnnnnnn nngctcttcc gatctgtaga yggatgggct 60
csgt 64

Claims (14)

1. A multiplex primer set, comprising: the kit comprises a group of upstream primers and a group of downstream primers, wherein the upstream primers are formed by connecting a joint 1, a primer bar code 1, a sequencing primer 1 and specific primers V1-V18 designed aiming at a variable region V region in series; the downstream primer is formed by connecting a joint 2, a primer bar code 2, a sequencing primer 2 and specific primers Va, Vd, Ve, Vg and Vm designed aiming at conserved sequences of C regions of gene constant regions of IgA, lgD, IgE, IgG and IgM in series; the sequences of the primer bar code 1 and the primer bar code 2 are different; the sequencing primer 1 and the sequencing primer 2 have the same sequence;
the specific primers V1-V18 in the upstream primer are respectively:
V1：5′-GCTGGGTGCGCCAGATGCCC
V2：5′-TGGATCCGTCAGCCCCCAGG
V3：5′-TGGATCCGTCAGCCCCCGGG
V4：5′-GTGCGACAGGCCCCTGGACAA
V5：5′-GTYCCGCAGGCCCCCGSACAA
V6：5′-GGGTGMGACTCGCTCGRGGACAA
V7：5′-GTGGCACAYGCCCCCGGACAA
V8：5′- GTGCGACAGGCWCGGAGACAA
V9：5′-TCCGCCAGCKCGGAGGGAAGG
V10：5′-TCCGRCTGCCCGCYCCGAA
V11：5′-TCCGGCAGMCGCCCCGGAA
V12：5′-TCCGGCAGCCCGCTCYGAAGG
V13：5′-GMAGCGCCAGSCTCCAGGGAA
V14：5′-GGAGKGCCAGGCTTCSGGGAA
V15：5′-GAAGCGCCWGGCTCCAGGGAA
V16：5′-GGAGCGCCAGGCTCCAMGGAA
V17：5′- GGAGCGCRAGGCTCCGGGCAA
V18：5′-GGAGCGCCAGCCTCYAGGGAA ；
specific primers Va, Vd, Ve, Vg and Vm in the downstream primer are respectively as follows:
Vg：5-ATGAYCGATGGGCCCTTGT
Va：5-GCAGACCTTRGGGCTGGTCA
Vm：5-GCGAATTCTCACWGGAGACG
Vd：5-GCGTGSCTGCAGCCTGATA
Ve：5-GTAGAYGGATGGGCTCSGT；
the upstream primer group of the multiplex PCR primer group is formed by mixing the following 18 upstream primers:
1：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGCTGGGTGCGCCAGATGCCC
2：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TTGGATCCGTCAGCCCCCAGG
3：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TTGGATCCGTCAGCCCCCGGG
4：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGTGCGACAGGCCCCTGGACAA
5：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGTYCCGCAGGCCCCCGSACAA
6：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGGGTGMGACTCGCTCGRGGACAA
7：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGTGGCACAYGCCCCCGGACAA
8：5′- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGTGCGACAGGCWCGGAGACAA
9：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TTCCGCCAGCKCGGAGGGAAGG
10：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TTCCGRCTGCCCGCYCCGAA
11：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TTCCGGCAGMCGCCCCGGAA
12：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TTCCGGCAGCCCGCTCYGAAGG
13：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGMAGCGCCAGSCTCCAGGGAA
14：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGGAGKGCCAGGCTTCSGGGAA
15：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGAAGCGCCWGGCTCCAGGGAA
16：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGGAGCGCCAGGCTCCAMGGAA
17：5′- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGGAGCGCRAGGCTCCGGGCAA
18：5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN（NN）GCT CTT CCG ATC TGGAGCGCCAGCCTCYAGGGAA；
wherein NNNN (NN) in the upstream primer group represents primer barcode 1;
the downstream primer group is formed by mixing the following 5 downstream primers:
1：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN（NN）GCT CTT CCG ATC TATGAYCGATGGGCCCTTGT
2：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN（NN）GCT CTT CCG ATC TGCAGACCTTRGGGCTGGTCA
3：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN（NN）GCT CTT CCG ATC TGCGAATTCTCACWGGAGACG
4：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN（NN）GCT CTT CCG ATC TGCGTGSCTGCAGCCTGATA
5：5‘-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN（NN）GCT CTT CCG ATC TGTAGAYGGATGGGCTCSGT；
wherein NNNN (NN) in the downstream primer represents primer barcode 2.
2. The multiplex primer set of claim 1, wherein: linker 1 sequence AAT GAT ACG GCG ACC ACC GAG ATC TAC AC; linker 2 sequence CAA GCA GAA GAC GGC ATA CGA GAT.
3. The multiplex primer set of claim 1, wherein: the sequences of sequencing primer 1 and sequencing primer 2 are GCT CTT CCG ATC T.
4. The multiplex primer set of claim 1, wherein: the primer bar code 1 and the primer bar code 2 consist of 6 or 8 nucleotides, and at least one nucleotide difference exists between the primer bar code 1 and the primer bar code 2 of different samples.
5. A method for constructing a human B cell immune repertoire based on high throughput sequencing using the multiplex primer set of claim 1, comprising the steps of:
1) extracting sample nucleic acid DNA or RNA;
2) carrying out first round PCR amplification on sample nucleic acid DNA or RNA to obtain a PCR amplification product 1;
3) recovering a PCR amplification product 1;
4) taking the recovered product of the PCR amplification product 1 as a template to carry out second round of PCR amplification to obtain a PCR amplification product 2;
5) recovering the PCR amplification product 2, wherein the recovered product forms a BCR sequencing library;
6) library detection:
the purity and the size of the obtained BCR sequencing library are detected by an Aglilent 2100 Bioanalyser; determining the concentration of the BCR library by using Nanodrop one;
7) high-throughput sequencing:
sequencing the purified BCR library by using Hiseq2500 of Illumina company, wherein the sequencing mode is PE 150;
8) and (3) data analysis:
after the sequencing data are filtered to remove the sequencing background, V \ D \ J gene comparison analysis is carried out on the off-line data and IMGT immune cell receptor database data, and a B cell immune repertoire spectrum is established.
6. The method for constructing a human B cell immune repertoire based on high-throughput sequencing according to claim 5, wherein when the sample is DNA, the PCR amplification system of the first round of PCR amplification in step 2) is formulated as follows: 5 XPCR Buffer 5.0 muL, dNTP Mix final concentration 0.5-1mM, multiple PCR primer group upstream primer group 3-5 muM, downstream primer group 3-5 muM, Mg2+The final concentration is 3-4mM, the Taq enzyme is 1.5-3unit, the DNA sample is 200-800ng, and DEPC water is added to make up the volume to 25 muL.
7. The method for high-throughput sequencing-based construction of human B-cell immune repertoire according to claim 6, wherein the amplification procedure of the first round of PCR amplification is as follows:
activating the hot start enzyme at 95 ℃ for 10-20 min; denaturation at 94-95 deg.C for 15sec, annealing at 55-60 deg.C for 60-120sec, and extension at 72 deg.C for 30-40sec, and performing 6-10 cycles; denaturation at 94-95 deg.C for 15sec, annealing at 65-70 deg.C for 40-60sec, and extension at 72 deg.C for 30-40sec, and performing 10-20 cycles; storing at 16 ℃.
8. The method for constructing a human B cell immune repertoire based on high-throughput sequencing according to claim 5, wherein, when the sample is RNA, the PCR amplification system of the first round of PCR amplification in step 2) is formulated as follows: 5 xRT-PCR Buffer 5.0 muL, dNTP Mix final concentration 0.5-1mM, multiple PCR primer group upstream primer group 3-5 muM, downstream primer group 3-5 muM, RNase inhibitor 0.25 muL, Mg2+The final concentration is 3-4mM, the mixture of reverse transcriptase and Taq enzyme is 2.0-3.5unit, the RNA sample is 200-.
9. The method for high-throughput sequencing-based construction of human B-cell immune repertoire according to claim 8, wherein the first round of PCR amplification is performed by the following amplification procedure:
reverse transcription is carried out for 35-40min at the temperature of 45-50 ℃; activating the hot start enzyme at 95 ℃ for 10-20 min; denaturation at 94-95 deg.C for 15sec, annealing at 55-60 deg.C for 60-120sec, and extension at 72 deg.C for 30-40sec, and performing 6-10 cycles; denaturation at 94-95 deg.C for 15sec, annealing at 65-70 deg.C for 40-60sec, and extension at 72 deg.C for 30-40sec, and performing 10-20 cycles; storing at 16 ℃.
10. The method for constructing a human B-cell immune repertoire based on high-throughput sequencing according to claim 5, wherein in step 3) and step 5), the PCR amplification product 1 and the PCR amplification product 2 are recovered and purified by agarose gel electrophoresis, purification column purification or magnetic bead purification.
11. The method for constructing a human B-cell immune repertoire based on high-throughput sequencing according to claim 5, wherein in step 4), the PCR amplification system for the second round of PCR amplification is formulated as follows:
5 xPCR Buffer 5.0 muL, dNTP Mix final concentration 0.2-0.4mM, linker 1 final concentration 0.2-0.5 muM, linker 2 final concentration 0.1-0.2 muM, Mg2+The final concentration is 2-2.5mM, the Taq enzyme is 1.5-2unit, the PCR amplification product 1 is recovered to obtain 5 muL, and DEPC water is added to complement the volume to 50 muL.
12. The method for high-throughput sequencing-based construction of a human B-cell immune repertoire according to claim 11, wherein the amplification procedure of the second round of PCR amplification is as follows:
activating the hot start enzyme at 95 ℃ for 10-20 min; denaturation at 94-95 ℃ for 10-20 sec, annealing at 50-55 ℃ for 30-40sec, extension at 72 ℃ for 30-40s, performing 30-40 cycles, and storing at 16 ℃.
13. The method for constructing the human B cell immune repertoire based on the high-throughput sequencing as claimed in claim 6 or 8, wherein the upstream primer group is formed by equimolar mixing of 18 upstream primers; the downstream primer group is formed by mixing 5 downstream primers in an equimolar way.
14. The method for constructing the human B cell immune repertoire based on high throughput sequencing according to claim 6 or 8, wherein the total number of moles of the upstream primer set is the same as the total number of moles of the downstream primer set.
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