Method for constructing immune repertoire high-throughput sequencing library for removing chimera sequence in sample, group of primers and kit

A technology of immune repertoire and sequencing library, applied in primers and kits and its application in next-generation sequencing, and in the field of preparation of high-throughput sequencing library of immune repertoire, which can solve the problem that the antibody sequence cannot be clearly quantified and removed, and cannot be obtained from Distinguish issues such as chimeric antibody sequences on the sequence, to achieve the effect of correcting bias and accurate immune repertoire information

Pending Publication Date: 2022-02-01
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some studies have shown that chimeric antibody sequences in samples formed by the amplification of a small number of known sequences can be identified by using sequence alignment, the extremely diverse and highly similar germline V(D)J genes produced by somatic recombination Similar antibodies, resulting in the inability to sequence chimeric antibody sequences from authentic antibody sequences
Although the negative impact of chimeric antibody sequences on the analysis of antibody repertoire characteristics and the search for high-affinity antibodies has been widely concerned, it is still not possible to clearly quantify and remove in-sample chimeric antibody sequences in Rep-seq data

Method used

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  • Method for constructing immune repertoire high-throughput sequencing library for removing chimera sequence in sample, group of primers and kit
  • Method for constructing immune repertoire high-throughput sequencing library for removing chimera sequence in sample, group of primers and kit
  • Method for constructing immune repertoire high-throughput sequencing library for removing chimera sequence in sample, group of primers and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Obtaining sample RNA

[0040] 1. Human peripheral blood was collected using EDTA anticoagulant tubes, and human peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation;

[0041] 2. Use human naive B cell sorting kit (Stemcell, 17254) to sort and obtain human naive B cells

[0042] 3. Count human peripheral blood mononuclear cells and naive B cells and extract total RNA (Qiagen, 74106). The concentration is shown in Table 7.

[0043] Table 7

[0044]

Embodiment 2

[0045] Example 2: Human BCR repertoire library construction

[0046] Using the total RNA obtained in Example 1 as a template, use the BCR subtype-specific primers with downstream Barcode and UMI to synthesize the first strand of cDNA by reverse transcription, and then use the leader region and V gene-specific primers with upstream Barcode and UMI The mixture is subjected to 1 cycle of PCR to realize UMI that is specifically paired with each template molecular marker in the sample, and then the upstream and downstream Barcode primers are used for unbiased amplification, as shown in the schematic diagram figure 1 , the specific process is as follows:

[0047] 1. cDNA synthesis by reverse transcription

[0048] Reverse transcription was performed using BCR subtype-specific primers (SEQ ID NO: 11-15, 23 and 35) with downstream Barcode and UMI, as follows:

[0049] a. Configure the mixed system according to Table 8, and carry out the reaction on the PCR machine. The reaction prog...

Embodiment 3

[0085] Example 3: Removing chimeric sequences in human naive B cell antibody repertoire sequencing data

[0086]The chimera sequence in the human naive B cell antibody library sequencing data in Example 2 was removed through the bioinformatics analysis process, specifically as follows Figure 4 .

[0087] 1. The off-machine data is identified and removed from the chimeric antibody sequences between samples by pairing Barcode;

[0088] 2. Based on the abundance distribution of UMI pairs, remove the chimeric antibody sequences in the samples that obey the poisson distribution;

[0089] 3. Re-cluster and establish consensus sequences based on CDR3s and UMI pairs, eliminate base errors introduced in PCR and high-throughput sequencing, and obtain real antibody sequences.

[0090] The amount of sequencing data before and after removing chimeras and constructing consensus sequences is shown in Table 15.

[0091] Table 15

[0092]

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Abstract

The invention provides a method for constructing an immune repertoire high-throughput sequencing library for removing a chimera sequence in a sample, a group of primers and a kit. Based on a multiplex PCR library preparation method, a set of primers is designed for each of a heavy chain and a light chain of BCR, and the primers can amplify all different alleles of IgG, IgM, IgA, IgD, IgE, IgK and/or IgL found at present. According to the method, 3'-terminal UMI is added in a reverse transcription process, then 5'-terminal UMI is added in a first round of PCR process, so that two ends of each amplified template molecule are marked by uniquely paired UMI sequences, and then a specific outer Barcode primer is used for carrying out second round of PCR amplification. According to the sequencing data obtained by constructing the library by using the method, the sample chimera sequence in the high-throughput sequencing data can be discriminated and removed by using the abundance distribution of the paired UMI in the analysis process, so that more accurate immune repertoire data can be obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing an immune repertoire high-throughput sequencing library with high accuracy, a set of primers and a kit and their application in next-generation sequencing. Background technique [0002] B cells are important executors of humoral immunity, and their cell membrane surface receptor BCR and secreted antibodies are important molecules that perform specific immune functions. The collection of all BCRs and antibodies within an individual or tissue is called the antibody repertoire. Antibody repertoires are very large and complex. During the development of B cells, the recombination of the germline V(D)J gene and the insertion or deletion of bases in the V-D and D-J junction regions endow the antibody repertoire with extremely high diversity, forming unique complementarity-determining regions that recognize various antigens3 BCR of naive B cells (CDR3). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C12Q1/6858C40B50/06C12N15/10C12N15/11
CPCC12Q1/6806C12Q1/6869C12Q1/6858C12N15/1096C40B50/06C12Q2600/16C12Q2525/191C12Q2531/113C12Q2537/143C12Q2535/122
Inventor 张镇海余学清唐海培曾惠昆汪启龙朱燕陈青云蓝春红关俊杰谢文汐
Owner GUANGDONG GENERAL HOSPITAL
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