Method for simultaneously sequencing multi-sample CDR3 (complementary determining region 3) receptor library with high flux

A high-throughput, multi-sample technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high sample preparation cost, cumbersome operation, heavy workload, etc., and achieve high-throughput sequencing cost reduction , the effect of extensive application value

Inactive Publication Date: 2012-05-09
ZUNYI MEDICAL UNIVERSITY
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Problems solved by technology

[0004] However, when using high-throughput sequencing technologies such as the Roche 454 sequencing platform and Illumina's solexa sequencing platform to sequence the CDR3 receptor library, different upstream and downst...

Method used

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  • Method for simultaneously sequencing multi-sample CDR3 (complementary determining region 3) receptor library with high flux
  • Method for simultaneously sequencing multi-sample CDR3 (complementary determining region 3) receptor library with high flux
  • Method for simultaneously sequencing multi-sample CDR3 (complementary determining region 3) receptor library with high flux

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Embodiment Construction

[0013] The technical solution of the present invention will be described in detail below in conjunction with the examples.

[0014] Take high-throughput sequencing analysis of the human TCR β chain CDR3 receptor library as an example:

[0015] 1. The human TCR β chain V gene family contains 45 functional gene families and 7 ORFs, and 36 upstream primers are designed according to the homology of 52 genes (see Table 1, 45 or 52 or other similar primers can also be designed source family combination).

[0016] 2. Primer design of multiple base compositions of the C and J genes of the human TCR β chain:

[0017] 1) According to the C gene of human TCR β chain (TRBC1 / TRBC2, highly homologous, see Table 2), multiple downstream primers suitable for pairing with upstream primers can be designed at the front end of the TRBC gene, such as:

[0018] (1) TRBC ttctgatggc tcaaacac;

[0019] (2) TRBC gaagcagag atctcccac;

[0020] (3) TRBC acctgaacaa ggtgttccca;

[0021] (4) TRBC ccacacc...

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Abstract

The invention discloses a method for simultaneously sequencing a multi-sample CDR3 (complementary determining region 3) receptor library with a high flux, comprising the following steps of: only designing one set of V-gene family upstream primers in the variable region of a TCR (T cell antigen receptor) or BCR (B cell antigen receptor) chain according to the homology of the V-gene family, then designing C or J gene characteristic downstream primers of the TCR or BCR chain as many as samples and suitable for being paired with the upstream primers, amplifying each sample by one characteristic downstream primer and the same set of upstream primers to obtain a CDR3 region of the TCR or BCR, then mixing all the amplified samples to be a to-be-sequenced sample library, finally sequencing, wherein the characteristic downstream primers are designed by different ATCG basic group compositions. The CDR3 sequences of multiple samples can be simultaneously sequenced by operating one high-flux sequencing program via the method for simultaneously sequencing a multi-sample CDR3 receptor library with a high flux disclosed by the invention, so that the high-flux sequencing cost is greatly decreased. Thus, a high-flux sequencing technology has a wide application value in a sequencing research of a CDR3 receptor library.

Description

technical field [0001] The invention relates to a sequencing method for a T / B cell CDR3 receptor library, in particular to a method for simultaneously sequencing multiple CDR3 receptor libraries with high throughput. Background technique [0002] In the past two decades, the TCR / BCR CDR3 receptor library of T / B lymphocytes has been used in the development, evolution, differentiation, proliferation and maturation of T / B cells, research on subgroups, and the relationship between T / B cells and infection, tumors, and autoimmunity. It has been widely used in the research of diseases, stem cells and organ transplantation. CDR3 receptor library research technology is based on the TCR / BCR gene rearrangement rules, set upstream primers in the variable region (variable) genes of TCR (alpha / beta / gamma / delta chain) and BCR (heavy / light chain), and connect Set downstream primers in the joining J gene or the constant C gene, amplify the PCR product of the target chain by PCR, and use SSC...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 姚新生贺晓燕
Owner ZUNYI MEDICAL UNIVERSITY
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