NGS targeted capture method based on dark probe technology and application of NGS targeted capture method in differential depth sequencing

A probe and sequencing technology, applied in the field of high-throughput sequencing library preparation, can solve the problem of not being able to meet the high depth of exon region sequencing at the same time

Pending Publication Date: 2022-05-27
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

WGS cannot simultaneously satisfy the high depth of sequencing in the exon region and the appropriate sequencing depth in the SNP locus region

Method used

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  • NGS targeted capture method based on dark probe technology and application of NGS targeted capture method in differential depth sequencing
  • NGS targeted capture method based on dark probe technology and application of NGS targeted capture method in differential depth sequencing
  • NGS targeted capture method based on dark probe technology and application of NGS targeted capture method in differential depth sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Preparation of all-exon and SNP site liquid capture probes required for WGS

[0043] 1. The human all-exon probe was QuarXeq Human AllExon Probes 3.0 from Shanghai Diying Biotechnology Co., Ltd., product number Y1009A (biotin-labeled), hereinafter referred to as WES. The target region of the WES probe is to use the NCBI Ref-Seq data to cover all the known exon regions.

[0044] 2. The human SNP probe was QuarXeq Human SNP Probes1.0 from Shanghai Diying Biotechnology Co., Ltd., the product number is Y1011A. The probe is a capture probe that covers 200,000 single nucleotide polymorphism sites (SNP) after cross-analysis using multiple Genome-wide association study (GWAS) project databases, and includes standard biotin-labeled probes As with dark probes without biotin labeling (identical probes, the only difference is whether they are labeled with Botin or not), probes labeled with Biotin are hereinafter referred to as SNP probes.

Embodiment 2

[0045] Example 2. Concentration adjustment and ratio of WES probe

[0046] The two types of probe groups in Example 1 were mixed according to different concentrations and ratios. Finally, the probes required for WES were prepared (WES probes were at a fixed concentration, the concentration of SNP probes and the ratio of biotin labeling were adjusted, the final product, WES probes used 300ng, SNPs (including probes with and without biotin labeling) The total amount is 100ng), the operation is as follows:

[0047] For the optimization of the dark probe competition binding scheme for the WES+SNP part, take 3 μL of the biotin-labeled SNP probe stock solution (50ng / μL), add 3 μL of the SNP dark probe without biotin labeling, and mix well. Named SNP-Dark2x, take 3 μL of SNP-Dark2, add 3 μL of SNP dark probe without biotin label, and name it SNP-Dark4x after mixing. Take 3 μL of SNP-Dark4, add 3 μL of SNP dark probe without biotin label, and name it SNP-Dark8x after mixing. Take 3...

Embodiment 3

[0048] Example 3. Liquid phase hybridization capture and sequencing of binary combined probes (WES+SNP, WES+SNP-Dark4x, WES+SNP-Dark8x, WES+SNP-Dark16x)

[0049] 1. Take 200ng NA12878 DNA human gene standard (a human genomic DNA standard, Coriell Institute) for library construction and probe test optimization. The kit used for library construction is QuarPrep Ultra DNALibrary Kit, item number L1001A, Shanghai Diying Biotechnology Co., Ltd. company.

[0050] 2. Start the Covaris S220 system (an ultrasonic DNA fragmenter): make sure that the fresh deionized water exceeds the level 12 on the Covaris tank; pre-cool and degas for half an hour. Add 200ng NA12878 DNA human gene standard, and make up the volume to 50μl with fresh deionized water, then add it to the Covaris micro Tube, stick to the wall and slowly pour out the liquid, being careful not to create air bubbles at the bottom of the tube.

[0051] 3. The ultrasonic conditions on Covaris S220 or M220 are set as shown in Tab...

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Abstract

The invention discloses an NGS targeted capture method based on a dark probe technology and application of the NGS targeted capture method in differential depth sequencing. The invention provides a targeted capture high-throughput sequencing method for simultaneously detecting exons and SNP sites in a target region range of a whole genome. The method comprises the following steps: designing two groups of original probes capable of capturing all exons and all SNP sites in the target region according to a nucleotide sequence of the target region; one of the two groups of original probes has a labeled form and an unlabeled form at the same time, and the other group only has a labeled form; matching and combining the two groups of probes according to different proportions of the labeled probes, and hybridizing with a genome library to be detected to obtain a capture library; and performing high-throughput sequencing. Compared with a standard exon sequencing method, the dark probe capture method disclosed by the invention has the advantages that the sequencing coverage degree is controlled according to the requirements of different areas under the same single-tube reaction, and the data utilization rate of NGS is remarkably improved. The dark probe method disclosed by the invention can be used as a high-cost-performance alternative scheme of the WGS.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a NGS target capture method based on dark probe technology and its application in differential deep sequencing, in particular to high-throughput sequencing library preparation, different target regions of exons and SNP sites Probe mix ratios, differential capture and sequencing methods in single-tube reactions. Background technique [0002] Currently, whole-exome high-throughput sequencing and genotyping microarrays are widely used in the research of complex diseases and various genetic diseases. Especially for GWAS-related complex disease research, SNP genotyping array and whole exome sequencing both play an important role. Genome-wide association study (GWAS) analysis method using SNP genotyping chip has made unprecedented achievements in the screening of susceptibility genes for complex diseases. However, since most of the SNPs on the chip are located in non-coding regions, and GW...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12N15/11C12Q1/6806C12Q1/6886C12Q1/6883
CPCC12Q1/6869C12Q1/6806C12Q1/6886C12Q1/6883C12Q2600/156C12Q2535/122C12Q2563/131C12Q2563/143C12Q2563/149
Inventor 师咏勇张满仓
Owner SHANGHAI JIAO TONG UNIV
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