Detection method for low-frequency mutation of circulating tumor DNA

Abstract

The invention discloses a detection method for low-frequency mutation of circulating tumor DNA (which is called as ctDNA for short in following). The method comprises the following steps: S1, extracting cfDNA from blood plasma; S2, repairing the tail end of the extracted cfDNA and adding A at the 3' end; S3, performing connector connection on a tail end repaired product, wherein a connector contains a random molecular tag sequence and contains an index sequence for distinguishing different samples; S4, performing PCR amplification on a connector connection product; S5, obtaining a target library through a probe; S6, sequencing through illumina Miseq, illumina Nextseq, illumina Hiseq platforms and analyzing offline data. The method is suitable for probe target capturing sequencing, can reduce an offline data repetition rate, can remove PCR and errors generated in a sequencing process, can improve ctDNA detection flexibility and specificity and can reduce a false positive rate.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing, in particular to a method for detecting low-frequency mutations in circulating tumor DNA. Background technique [0002] cfDNA refers to a kind of extracellular DNA existing in blood and cerebrospinal fluid in the form of single-stranded or double-stranded DNA or DNA-protein complexes. In 1948, Mandel and Metais first discovered the presence of cell-free DNA in human blood. In tumor patients, the free DNA fragments released into the blood after tumor cell necrosis and apoptosis are called circulating tumor DNA (ctDNA). The size of ctDNA fragments is generally about 160-180bp, and the half-life is 16min-2h. [0003] Because ctDNA carries all the mutation information of tumor cells, and these gene mutations only exist in the genome of precancerous cells or cancer cells, and will not appear in the DNA of other normal cells in the same body, ctDNA is relatively different from traditional hi...

AI Technical Summary

Problems solved by technology

However, the biggest disadvantage of this method is that its principle is to build a library by multiplex PCR, so it can only detect a small number of regions or sites, and the detection range is far less than that of probe capture sequencing technology.

In addition, the current general-purpose molecular tags are randomly combined with four bases of ATCG, and the length is generally 8-12bp. Theoretically, the four bases are completely randomly distributed. The required energy and synthesis efficiency are different, resulting in the frequency of ATCG appearing at each location is not exactly the same

There may be multiple consecutive identical bases, such as 8 A or C, so that the actual number of molecular labels obtained is less than the theoretical value

Moreover, multiple consecutive identical bases will also increase the possibility of sequencing errors and increase the proportion of dominant molecular sequences

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