Low-quality FFPE DNA processing method and device and storage medium

Abstract

The invention discloses a low-quality FFPE DNA processing method and device and a storage medium. The processing method comprises the following steps: carrying out fragmentation treatment on FFPE DNA,performing heating to remove a secondary structure, and carrying out enzyme digestion; repairing the tail end after digestion, adding poly A tail, and using an A base connector; then, carrying out PCR amplification, hybrid capture and sequencing to obtain sequencing data; and comparing the sequencing data with a human reference genome, and filtering out sequences which are partially compared to two different places of the same chromosome at the same time and of which the comparison initial position distance is less than 500bp from a comparison result. According to the method disclosed by theinvention, most of single chains are eliminated through the heating and the enzyme digestion, so that the single chains cannot form stable double chains in terminal filling-in after being connected toone another, and false positive of subsequent mutation detection caused by single chain residues is reduced; and through data comparison and filtering, single-chain interference is further removed, and the accuracy of mutation detection results is improved.

Description

technical field [0001] This application relates to the field of FFPE sample processing, in particular to a processing method, device and storage medium for low-quality FFPE DNA. Background technique [0002] Formalin-Fixed and Paraffin-Embedded (Formalin-Fixed and Paraffin-Embedded, abbreviated as FFPE) samples, that is, FFPE samples. Formalin-fixed paraffin embedding is a routine method of preservation of tissue samples that has been used clinically and scientifically for a century. The huge number of archived FFPE samples is valuable experimental material for retrospective studies, elucidation of disease mechanisms, discovery of therapeutic targets, and indication of prognosis. The DNA in FFPE samples (hereinafter referred to as FFPE DNA) is prone to cross-linking, high-temperature wax immersion, high-temperature de-crosslinking, etc. after formalin treatment, and after long-term storage, it is prone to severe chain breakage, single-strandization, and loss of DNA. Base s...

AI Technical Summary

Problems solved by technology

However, the single-strand residues, stem-loop structures, and dimer products of complementary pairing in the single-strand region after interruption in low-quality FFPE DNA samples will not be eliminated during the end repair process of these products, so they continue to enter the construction process. Library sequencing, and then in the sequencing results, Watson and Crick chain information appear in the forward + reverse sequence chimerism of a read at the same time, resulting in a large number of false positive sites in the mutation detection

None of the existing library construction kits or library construction methods provide a complete solution, nor do they give suggestions on how to eliminate this effect

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