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Design method of nucleic acid capture probe for HLA typing

A nucleic acid capture and HLA-A technology, applied in the design of probes, nucleic acid capture probes for HLA typing and their design fields, can solve the problems of optimization, large differences, and failure to take into account, and achieve the effect of efficient capture

Active Publication Date: 2020-02-28
上海仁东医学检验所有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

In addition, the INDEL of small fragments has not been considered in the existing technology. The GC ratios of the Exon2 and Exon3 regions of the HLA genes are both above 60%, which is quite different from other regions on the human genome. The existing technology does not because of this difference. There are experimental optimizations

Method used

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  • Design method of nucleic acid capture probe for HLA typing
  • Design method of nucleic acid capture probe for HLA typing
  • Design method of nucleic acid capture probe for HLA typing

Examples

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Effect test

Embodiment 1

[0030] Example 1 Design of nucleic acid capture probes for HLA typing

[0031] 1) According to the full lengths of the known subtypes of HLA genes (the length of most HLA genes is 3503bp), construct the sequence libraries of HLA-A, HLA-B, and HLA-C respectively.

[0032] 2) Use DECIPHER (from the R package of bioconductor) to perform multiple sequence alignments to find the main SNP sites on Exon2 and Exon3 of HLA-A, HLA-B, and HLA-C genes and within 100 bp upstream and downstream.

[0033] Taking Exon2 of the HLA-A gene as an example, the specific comparison steps are as follows:

[0034] ① Load the DECIPHER package——library(DECIPHER);

[0035] ②Use readDNAStringSet to read the fasta file of HLA-A——hla_A.fa;

[0036] ③Use the AlignSeqs function to align the read sequences;

[0037] ④Use writeXStringSet to write the aligned DNA sequence into a new fasta file, and the new fasta file is the result of MSA.

[0038] After alignment, a Multiple Sequence Alignment (MSA) alignmen...

Embodiment 2

[0069] Example 2 Nucleic acid capture effect verification

[0070] DNA was extracted from 23 normal tissue samples, and the probes designed in Example 1 above were used for hybridization and capture of HLA genes. The library construction kits Accel-NGS ® 2S Hyb DNA Library Kit (Cat. No. 23024 / 23096) and 2S Set A / B Indexing Kit (Cat. No. 26148 / 26248) from Swift Company were used for library construction.

[0071] The operation steps of hybrid capture are as follows:

[0072] 1. gDNA fragmentation and purification

[0073] 1) Take 500ng gDNA according to the concentration of Qubit, add water to make up to 100μl, add covaris 130μl interrupted tube, set the program: 50W, 20%, 200 cycles, 330s. Take 1 μl after the interruption, and use the Qsep100 automatic nucleic acid and protein analysis system to detect the fragment distribution, and the main peak is 150-200 bp.

[0074] 2) Transfer the interrupted product to a new 1.5ml centrifuge tube, add 1.4 times the volume of AMPure b...

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Abstract

The invention discloses a design method of a nucleic acid capture probe for HLA typing. The design method comprises the following steps: 1) constructing HLA-A, HLA-B and HLA-C sequence libraries; 2) performing multiple sequence alignment, and searching SNP sites on each gene Exon2 and Exon3 and upstream and downstream SNP sites; 3) selecting an area covering a set number of SNP sites as a probe design candidate area by using a sliding window separation algorithm; 4) performing clustering analysis to obtain a representative sequence of each probe design candidate region as a candidate probe; 5)repeating all the candidate probes to obtain capture probes of the three genes. The invention also discloses the probe designed by the method, and the sequence of the probe is shown as SEQ ID NO: 9-88. The nucleic acid capture probe is designed for all polymorphic sites of Exon2 and Exon3 regions with the GC proportion of 60% or above in the HLA gene, and the hybrid capture effect of the HLA geneprobe is improved.

Description

technical field [0001] The present invention relates to the field of gene sequencing, in particular to the design of probes, more specifically, to nucleic acid capture probes for HLA typing and a design method thereof. Background technique [0002] High-throughput sequencing is also known as next-generation sequencing. In recent years, with the advancement of high-throughput sequencing technology, the cost of sequencing has been decreasing, the objects of sequencing services and application segments have continued to expand, and the market size of high-throughput sequencing has continued to increase. With favorable policy conditions, the clinical application of high-throughput sequencing technology in reproductive health and tumor personalized medicine has entered the fast lane, with broad application prospects. [0003] The sensitivity of NGS (next-generation gene sequencing) detection technology is much higher than that of the current traditional detection technology. It c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B25/20G16B30/10
CPCG16B25/20G16B30/10
Inventor 赵国栋乔宗赟
Owner 上海仁东医学检验所有限公司
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