A DNA low-frequency mutation enrichment sequencing method for exfoliated cells

Abstract

The present invention provides a low-frequency mutation enrichment sequencing method for exfoliated cell DNA, including the extraction of exfoliated cell DNA and DNA fragmentation, sample DNA library construction, general library TT-COLD PCR amplification enrichment, and probe enrichment capture , capture product PCR and on-machine sequencing, positive and negative double-strand error correction low-frequency information analysis steps, specifically TT COLD PCR based on joint universal primers to achieve first-level mutation enrichment and amplification for all types of mutations; design enrichment probe chip For hotspot mutations, replace the probe designed based on the human genome reference sequence hg19 with the probe designed based on the mutant base, and keep the probes at other sites unchanged, and perform the second-level enrichment capture; the two ends of the inserted DNA in the library construction are 12bp The self-sequence is used as a tag for positive and negative double-strand error correction comparison, which improves data utilization and realizes low-frequency accurate detection. It can detect 0.01% low-frequency variation with high specificity.

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...

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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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