Low-frequency mutations enrichment sequencing method for free target dna in plasma
AssigneeGeneplus - Beijing
InventorsXiaoxing Lv, Xin Yi, Meiru Zhao, Yanfang Guan, Tao Liu, Ling Yang
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Abstract
The present invention provides a low-frequency mutation enrichment sequencing method for free target DNA in plasma, comprising plasma DNA extraction and library construction, general library TT COLD PCR amplification enrichment, probe enrichment capture, PCR and sequencing of captured products, and positive and negatice double-strand error-correction low-frequency information analysis.
Claims21
A method for enrichment and sequencing of low-frequency mutations of target DNA in plasma, comprising the following steps:
- (1) extraction of the target DNA from plasma and library construction;
- (2) universal library TT-COLD PCR amplification and enrichment;
- (3) enrichment and capture with probes, and amplification and sequencing of hybridization capture products;
- and
- (4) analysis on low-frequency information with forward and reverse double-strand error correction.
A kit for enrichment and sequencing of free low-frequency mutation of target DNA in plasma, comprising an enrichment probe chip, the probes on the chip are as follows:
- a probe designed based on the human genome reference sequence hg19 is replaced with a probe designed based on a mutant base, the probes for other sites are not changed, and the difference ratio between the total coverage of the probe for the hotspot mutation and the coverage of normal probe for other regions is at least 3:1;
- the method for designing a probe based on a target DNA mutation base is set as follows:
- chip capture range is determined according to the purpose of the target gene, at least one most important hotspot mutation site is determined within a certain base range with reference to the database to which the target DNA belongs, several primary types of mutations among multiple mutation types present with respect to this site are taken for reference, corresponding frequency of occurrence is used as the proportion occupied by the mutation type in the total probe coverage level at the site.
A system for enrichment and sequencing of low-frequency mutation of ctDNA in plasma, comprising:
- (1) a library construction unit for ctDNA in plasma;
- (2) a universal library TT-COLD PCR amplification and enrichment unit;
- (3) a probe enrichment and capture unit, and an amplification and sequencing unit for hybridization capture products;
- and
- (4) an analytic unit for low-frequency information with forward and reverse double-strand error correction.
15-18. (canceled)
Dependencies are inferred from the claim text.