Method for low-frequency mutant-enriched sequencing of DNA of exfoliative cells

Abstract

The invention provides a method for low-frequency mutant-enriched sequencing of DNA of exfoliative cells. The method includes the steps of extraction of the DNA of the exfoliative cells, break of the DNA, sample DNA library construction, universal library TT-COLD PCR amplification enrichment, probe enrichment capture, captured product PCR and computer sequencing and positive-negative double-strand error-correction low-frequency information analysis, to be more specific, performing universal adapter primer based TT COLD PCR on all types of mutant to realize first-stage mutant-enriched amplification; designing an enrichment probe chip to replace a probe designed for a human genome reference sequence hg19 with a probe designed based on mutation bases for hot point mutants, keeping probes at other sites unchanged, and performing second-stage enriched capture. 12bp own sequences inserted into two ends of the DNA in library construction are taken as labels for positive and negative double-strand error correction comparison, data utilization rate is increased, low-frequency accurate detection is realized, and high-specificity detection for 0.01% low-frequency mutations can be achieved.

Description

technical field [0001] The invention belongs to the technical field of bioinformatics high-throughput sequencing, and in particular relates to a DNA low-frequency mutation enrichment sequencing method for exfoliated cells. Background technique [0002] Exfoliated cells refer to the inner surface mucosa of natural lumen organs. Under normal circumstances, the epithelial cells of human organ mucosa are often exfoliated and renewed. They are generally divided into three categories: natural exfoliated cells of nasopharynx, oral cavity, esophagus, vagina, etc., and some artificial cells. Cells obtained by brushing; body cavity aspirated fluid (pleural effusion, cerebrospinal fluid, pericardial effusion, etc.); needle aspiration cells. Due to its safety, easy operation of equipment, and based on its histopathological characteristics, a new discipline, exfoliated cytopathology, has gradually developed, which is widely used in the early detection and diagnosis of related tumors. Ho...

AI Technical Summary

Problems solved by technology

In addition, the overall traditional clinical detection and diagnosis process is still time-consuming and laborious, and a more accurate and practical detection method is urgently needed

[0003] At present, with the rapid development of molecular biology and sequencing technology, the detection of exfoliated cells based on high-throughput sequencing technology is gradually entering the clinic, especially the high-throughput sequencing technology based on HPV typing of cervical exfoliated cells is simple and fast , high-throughput, high-accuracy and other characteristics, is gradually replacing the traditional cervical Pap smear method, but the current conventional sequencing technology itself has a certain error rate, and due to individual differences, tumor development period, sampling operations and other reasons , the abundance of tumor cells in exfoliated cells often fluctuates greatly, even at a low abundance level of about 0.1%, resulting in certain false negatives and false positives based on conventional sequencing techniques

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