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HLA (human leucocyte antigen) typing method based on PacBio RS II sequencing platform

A typing method and sequencing platform technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as low precision, inability to test HLA genes, and inability to separate alleles, achieving ultra-high The effect of resolution

Active Publication Date: 2015-12-02
BEIJING GRANDOMICS BIOTECH
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Problems solved by technology

However, there are also problems with the second-generation sequencing technology, mainly because it is impossible to test all the HLA genes, or it is limited to exons 2, 3, and 4, and the sequences of introns and UTR regions cannot be obtained.
[0004] The number of HLA types continues to increase, reaching 12,242 (IMGT / HLA database), but the sequencing method is still limited to exons 2, 3, and 4, with low accuracy, and in many cases, alleles cannot be separated

Method used

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  • HLA (human leucocyte antigen) typing method based on PacBio RS II sequencing platform
  • HLA (human leucocyte antigen) typing method based on PacBio RS II sequencing platform
  • HLA (human leucocyte antigen) typing method based on PacBio RS II sequencing platform

Examples

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Embodiment 1

[0028] Example 1: DNA extraction, sequencing and HLA typing of 82 samples

[0029] In this example, DNA was extracted from oral mucosal cell samples, DNA fragments of HLA-A, HLA-B, and HLA-C were amplified, then mixed, sequenced with a PacBioRSII sequencing instrument, and finally HLA typing was performed.

[0030] 1. Sample collection: The oral mucosal cells are collected and preserved by a disposable sampling swab (registered product number is YZB / Guangdong A0278-2012, Shenzhen Mai Rui Kelin Technology Co., Ltd.), the preservation solution is 2 mL.

[0031] 2. DNA extraction: Qiagen's Blood&CellCultureDNAKit kit is used for extraction, the volume of the extracted liquid is about 80μL, and the product of one DNA extraction can do about 20 PCRs.

[0032] 3. PCR amplification: design primers in the 5'UTR and 3'UTR regions of the three genes of HLA-A, HLA-B and HLA-C, and add a barcode sequence to the 5'end of the primers. The barcode sequence is to distinguish the samples. Each sample ...

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Abstract

The invention discloses an HLA (human leucocyte antigen) typing method based on a PacBio RS II sequencing platform. A collected sample is subjected to DNA extraction and then to PCR (polymerase chain reaction) amplification, PCR products are mixed to establish a 10k library, and PacBio RS II sequencing is performed; then, original data obtained by sequencing are corrected, and HLA typing is performed with software programs. Compared with existing HLA typing methods, the HLA typing method has super-high resolution and is of important value to applications such as clinical graft tissue matching, population genetics, anthropology and evolutiology, as well as basic research work.

Description

Technical field [0001] The present invention relates to the technical field of gene sequencing, in particular to an HLA gene sequencing typing method, in particular to a method for typing full-length HLA-A, HLA-B, and HLA-C genes based on the sequencing of the third-generation sequencer PacBioRSII The method is mainly used to classify HLA genes with ultra-high resolution. Background technique [0002] The human leukocyte antigen (HLA) system is another name for the human major histocompatibility complex (MHC), and is the most immune-related genomic region in the human body. It is located on the short arm of human chromosome 6 and consists of a series of closely linked loci. HLA genes have the highest genetic polymorphism in the human genome, and the degree of HLA type differences between individuals is very large. HLA gene has the function of recognizing self and non-body and regulating immune response. In medicine, matching the correct and high-precision HLA type plays a deci...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2563/185C12Q2537/165
Inventor 梁德全汪德鹏马传艳
Owner BEIJING GRANDOMICS BIOTECH
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