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A HLA typing method based on pacbio RS II sequencing platform

A sequencing platform and typing method technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as low precision, inability to separate alleles, and inability to obtain sequence information, and achieve ultra-high resolution rate effect

Active Publication Date: 2019-01-25
BEIJING GRANDOMICS BIOTECH
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AI Technical Summary

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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  • A HLA typing method based on pacbio RS II sequencing platform
  • A HLA typing method based on pacbio RS II sequencing platform
  • A HLA 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, and then the samples were mixed, sequenced with a PacBio RSII sequencing instrument, and finally HLA typing was performed.

[0030] 1. Sample collection: Collect and preserve oral mucosal cells with a disposable sampling swab (registered product number YZB / Guangdong A0278-2012, Shenzhen Mairui Kelin Technology Co., Ltd.), and the preservation solution is 2mL.

[0031] 2. DNA extraction: Qiagen’s Blood&Cell Culture DNA Kit was used for extraction. The liquid volume after extraction was about 80 μL, and the DNA product extracted once could be subjected to 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 barcode sequences to the 5' ends of the primers. The barcode sequence is use...

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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 HLA-A, HLA-B, and HLA-C full-length genes based on the sequencing of the third-generation sequencer PacBio RSII The method is mainly used to classify HLA gene types with super high resolution. Background technique [0002] The human leukocyte antigen (Human leukocyte antigen, HLA) system is another name for the human major histocompatibility complex (MHC), and it is a genomic region most related to immunity 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 gene has the highest gene polymorphism in the human genome, and the HLA type difference between individuals is very large. HLA genes have the functions of identifying self and non-self, regulating immune response and so on. In medicine, the correct and high-prec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2563/185C12Q2537/165
Inventor 梁德全汪德鹏马传艳
Owner BEIJING GRANDOMICS BIOTECH
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