Second-generation sequencing method for dimolecular self-checking library preparation and hybrid capture used for trace DNA ultralow frequency mutation detection

A DNA molecule and DNA library technology, applied in the biological field, can solve problems such as inherent sequencing errors

Active Publication Date: 2018-01-19
SHANGHAI DYNASTYGENE CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, high-throughput sequencing instruments have inherent sequencing errors of 0.1%-1% (Illumina HiSeq 0.1%, ABI SOLiD 0.2%, Life Ion Torrent 1%)

Method used

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  • Second-generation sequencing method for dimolecular self-checking library preparation and hybrid capture used for trace DNA ultralow frequency mutation detection
  • Second-generation sequencing method for dimolecular self-checking library preparation and hybrid capture used for trace DNA ultralow frequency mutation detection
  • Second-generation sequencing method for dimolecular self-checking library preparation and hybrid capture used for trace DNA ultralow frequency mutation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Example 1. Preparation and purification of self-verifying bimolecular identification code hairpin adapter

[0149] The self-verifying bimolecular identification code hairpin connector of the present invention is as follows: figure 1 shown.

[0150] 1. Synthesis of linker primers

[0151] Design and synthesize the following four single-stranded DNA molecules: single-stranded DNA molecule A, single-stranded DNA molecule B, single-stranded DNA molecule C and single-stranded DNA molecule D, which are named DYMB-6a, DYMB-6b, and DYMB- 6c and DYMB-6d. The sequence is as follows:

[0152] DYMB-6a:

[0153]

[0154] DYMB-6b:

[0155]

[0156] DYMB-6c:

[0157]

[0158] DYMB-6d:

[0159]

[0160] Each single-stranded DNA molecule includes, from the 5' end to the 3' end, the protection sequence of the enzyme recognition site, the enzyme recognition site, the fixed spacer sequence, the random molecular tag sequence, the stem A (Illumina standard sequencing prim...

Embodiment 2

[0198] Embodiment 2, circulating cell-free DNA ultra-low frequency variation detection method

[0199] The experimental flow chart of the circulating cell-free DNA ultra-low frequency variation detection method of the present invention is as follows image 3 shown.

[0200] 1. Construction of library for cfDNA low-frequency mutation detection (DY-Ultra)

[0201] 1. End repair and A-tailing at the 3' end of circulating free DNA (cfDNA) in trace amounts of plasma

[0202] Take qualified cfDNA (cfDNA standard HD779 from Horizon Company, 0.1% mutation frequency, Multiplex I cfDNA Reference Standard, containing 8 known variations: L858R, ΔE746-A750, T790M, V769- D770insASV, G12D of the KRAS gene (Genebank ID 3845), Q61K and A59T of the NRAS gene (Genebank ID 4893), E545K of the PIK3CA (Genebank ID 5290) gene) total 30ng, diluted to 50uL with Low TE, and added 20uL of end repair solution (Wuxi Diying Biotechnology Co., Ltd., D8011A), and incubate at 20 degrees for 30 minutes to o...

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Abstract

The invention discloses a second-generation sequencing method for dimolecular self-checking library preparation and hybrid capture used for trace DNA ultralow frequency mutation detection. The methodcomprises the following steps: extracting plasma free DNA, performing DNA chemical error repair, preparing a self-checking dimolecular identification code hairpin type joint, repairing plasma free DNA, connecting DNA with the joint, and performing Pre-PCR amplification, excessive hybrid capture, Post-PCR amplification, computer sequencing, data error correction, and mutation analysis and annotation. The method disclosed by the invention can efficiently realize low frequency mutation detection of plasma free DNA. DNA error repair and dual redundancy checking technology can enable the method tohave ultralow false positive rate and high sensitity while detecting trace samples, thus avoiding defects of existing plasma circulating free DNA detection methods, not only realizing cancer mutationdetection and targeted medication guidance, but also realizing early screening of genetic and birth defects of fetus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the construction of a library for ultra-low frequency mutation detection of trace DNA (including: circulating free DNA, circulating tumor DNA, fetal free DNA, etc.) related to molecular biology, high-throughput sequencing technology and bioinformatics with targeted enrichment sequencing methods. Background technique [0002] Compared with traditional tissue biopsy, Liquid Biopsy has many advantages such as rapidity, convenience, and less damage. Among various liquid biopsy technologies, circulating cell-free DNA (ccfDNA) detection has developed rapidly due to its unique advantages and the maturity of high-throughput sequencing technology. In the human body, free DNA fragments from various sources flow into the blood circulation at all times, and free DNA fragments excreted from tumors of tumor patients and fetuses of pregnant women are also mixed in it. Circulating tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6827C12Q1/6869C40B40/06C40B50/06C12N15/11
Inventor 师咏勇周娟沈佳薇
Owner SHANGHAI DYNASTYGENE CO
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