Ultra-low-frequency library building method for FFPE tissue samples

Abstract

The invention discloses an ultra-low-frequency library building method for FFPE tissue samples. The ultra-low-frequency library building method for FFPE tissue samples comprises the following steps: DNA extraction of FFPE samples; DNA interruption; dephosphorylation and DNA denaturation; adding of biotin UID single-chain joints; capturing and elution of streptomycin magnetic beads; adding of double-chain second joints and elution of a library; adding of a second joint at the other end of a target DNA molecule by T4DNA ligase, and removal of excessive and residual joints by washing; LM-PCR amplification and introduction of barcode; and purification of a PCR amplification product by magnetic beads. Library building, capturing and sequencing are realized for some severely degraded FFPE DNA micro-samples, UID label joints are introduced before beginning of library building, and thus, preconditions are provided for follow-up exclusion of technical mutations introduced in library building, capturing and computerized sequencing process.

Description

technical field [0001] The invention relates to an ultra-low frequency library building method for FFPE tissue samples, which mainly solves the problem of library building and sequencing of FFPE samples in the NGS-based sequencing process. Background technique [0002] At present, there are two main types of database construction methods for FFPE samples: [0003] The first category is the method of double-stranded DNA library construction. The shortcomings of this method are mainly manifested in the following aspects: [0004] 1. The amount of double-stranded DNA required to build a library is large, so it cannot meet the needs of some clinical tissue samples, such as FFPE samples of punctured tissues, FFPE samples of 5-10 years, etc.; [0005] 2. Due to the multi-step purification process in the process of building the library, some short or ultra-short DNA, single-stranded DNA, and severely damaged DNA are all lost, which undoubtedly makes the already precious samples wo...

AI Technical Summary

Problems solved by technology

[0004] 1. The amount of double-stranded DNA required to build a library is relatively large, so it cannot meet the needs of some clinical tissue samples, such as FFPE samples of punctured tissues, FFPE samples of 5-10 years, etc.;

[0005] 2. Due to the multi-step purification process in the process of building the library, some short or ultra-short DNA, single-stranded DNA, and severely damaged DNA are all lost, which undoubtedly makes the already precious samples worse, resulting in Some ultra-low frequency mutations cannot be detected;

[0006] 3. Due to the need to perform end repair, PCR enrichment, and computer sequencing on some severely degraded samples during the library construction process, there is a possibility of introducing technical mutations in these processes, resulting in inaccurate detection

[0008] 1. During the single-stranded library construction process, the connection between the single-stranded adapter and the single-stranded DNA has a preference, and the connection efficiency is relatively low, which still requires a large initial sample size for library construction;

[0009] 2. In the existing single-stranded DNA library construction, there are still common problems with double-stranded DNA library construction, that is, there are also mutations introduced by human manipulation that cannot be ruled out