Method for high-throughput sequencing of tcr or bcr and method for correcting multiple pcr primer bias by using tag sequence

A tag sequence, high-throughput technology, applied in the field of molecular biology, can solve the problems of PCR primer amplification error, increase the complexity of primers, etc., and achieve the effect of reducing sequencing error, length and quantity

Inactive Publication Date: 2016-02-17
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when high-throughput sequencing technologies such as Roche454 sequencing platform and Illumina's solexa or GenomeAnalyzer sequencing platform are used for sequencing, library construction of samples before sequencing is generally required. Due to the diversity of TCR or BCR, the amplification preference of primers and the template The design of primers is particularly important when building a library due to factors such as differences in abundance; in addition, when building a library in the prior art, a tag sequence is often connected to the 5' end of the primer to track the specific target sequence, but the tag sequence makes the primer sequence extended. The complexity of the primers is increased. When multiple pairs of primers are used at the same time, ordinary PCR cannot meet the requirements of high-throughput sequencing library construction, and may cause amplification errors of PCR primers

Method used

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  • Method for high-throughput sequencing of tcr or bcr and method for correcting multiple pcr primer bias by using tag sequence
  • Method for high-throughput sequencing of tcr or bcr and method for correcting multiple pcr primer bias by using tag sequence
  • Method for high-throughput sequencing of tcr or bcr and method for correcting multiple pcr primer bias by using tag sequence

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Embodiment 1 of the present invention provides a method for preparing a B lymphocyte receptor (BCR) RNA sample, comprising the following steps:

[0076] (1) Collect 10 milliliters (ml) of fresh peripheral blood samples from healthy people;

[0077] (2) Using the principle of density gradient centrifugation (the density of Ficoll is 1.077, and the average density of PBMC is 1.072), and through repeated centrifugation and washing, relatively pure PBMCs are obtained. The steps are as follows:

[0078] a. Dilute the blood obtained in step (1) 1:1 with PBS or 0.09% NaCl;

[0079] b. Add 0.5 times the volume of the resulting diluted blood lymphocyte separation solution to the 15ml centrifuge tube;

[0080] c. Slowly add the diluted blood obtained in step a to the surface of the lymphocyte separation liquid obtained in step b to keep the separation liquid and blood layered, and then centrifuge with a swingangle centrifuge at a speed of 600g for 20min-25min at a lower speed, a...

Embodiment 2

[0085] Embodiment 2 of the present invention provides a method for using BCR full-length primers to perform two cycles of multiplex PCR to construct a BCR full-length high-throughput sequencing library for high-throughput sequencing, including the following steps:

[0086] (1) Reverse transcribe the total RNA obtained in Example 1 into cDNA, the method is as follows:

[0087] (1.1) Configure the following system on ice:

[0088]

[0089]

[0090]The BCR full-length downstream primers in this system are mIgG, mIgA, mIgM, mIgE or mIgD downstream primers (respectively shown in SEQ ID NO: 23 ~ SEQ ID NO: 27) and mixed with sequencing adapters, specifically as SEQ ID NO: 23 The 5' ends of the primers shown in ~SEQ ID NO:27 were respectively connected with the downstream primer adapter sequence of Illumina Sequencing Company and then mixed equimolarly. Throughput sequencing library construction instruction), the total RNA is the total RNA prepared in Example 1, and the water ...

Embodiment 3

[0114] Example 3 of the present invention provides a method of using CDR3 primers of BCR to perform two cycles of multiplex PCR to construct a high-throughput sequencing library in the CDR3 region of BCR for high-throughput sequencing. The full length of -V H 1. Full length-V H 2. Full length-V H 3. Full length-V H 4. Full length-V H 5. Full length-V H 6 and full length-V H The upstream primer of 7 was replaced by CDR3-V H 1. CDR3-V H 2. CDR3-V H 3. CDR3-V H 4. CDR3-V H 5. CDR3-V H 6 and CDR3-V H 7 upstream primers (as shown in SEQ ID NO: 28 to SEQ ID NO: 39), other operations and experimental conditions remained unchanged.

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Abstract

The invention provides a method for carrying out high-throughput sequencing on a TCR (T cell receptor) or a BCR (B cell receptor). The method is characterized by designing upstream primers according to gene features of a V region of the TCR or the BCR and designing downstream primers according to gene features of a C region or a J region of the TCR or the BCR and obtaining sequences of the of the TCR or the BCR in combination with the multiplex PCR (polymerase chain reaction) technology and high-throughput sequencing, thus analyzing the rearrangement information of the TCR or the BCR. Compared with 25-30 cycles of existing multiplex PCR, two cycles of the multiplex PCR technology provided by the invention can conduce to greatly reducing the sequencing errors caused by primer amplification preference. Besides, the invention also provides a method for correcting multiplex PCR (polymerase chain reaction) primer deviation by utilizing DNA (deoxyribonucleic acid) tag sequences, thus further reducing the sequencing errors caused by primer amplification preference and intrinsic sequencing errors of high-throughput sequencing.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for high-throughput sequencing of TCR or BCR and a method for correcting the bias of multiple PCR primers by using tag sequences. Background technique [0002] During the development and maturation of T or B lymphocytes, a large and diverse TCR or BCR receptor library is formed through V(D)J gene combination, junctional diversity, and somatic hypermutation, gene conversion, and class switching. , the diversity of TCRs in the immune cell population is as high as 10 16 , the diversity of BCR up to 10 14 , Traditional methods such as SSCP technology, GeneScan technology, fluorescent quantitative melting curve technology, etc., often have the disadvantage of narrow coverage range, which cannot meet the detection of a large number and variety. [0003] High-throughput sequencing can meet the huge sequence sequencing requirements. However, when high-throughput s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2537/143C12Q2535/122C12Q2563/185
Inventor 徐莹李周芳童寅张萌葛良进贺建奎
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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