Detection method for low-frequency mutation of circulating tumor DNA

A detection method and low-frequency mutation technology, applied in the field of high-throughput sequencing, can solve the problems of increasing the proportion of dominant molecular sequences, the number of molecular labels is small, and the frequency is not exactly the same, so as to reduce the data repetition rate, avoid consecutive bases, and save The effect of data volume

Active Publication Date: 2018-11-23
厦门基源医疗科技有限公司
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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. ...

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  • Detection method for low-frequency mutation of circulating tumor DNA
  • Detection method for low-frequency mutation of circulating tumor DNA
  • Detection method for low-frequency mutation of circulating tumor DNA

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Embodiment 1

[0030] 10ml of whole blood was collected from 5 patients with lung cancer, and the collection tubes were Streck Cell-Free DNA BCT® BloodCollection Tubes (10ml), stored and transported at room temperature for no more than 72 hours. Use two-step method to separate plasma. First, centrifuge the whole blood at 1600g for 10min at room temperature, take out about 4.5ml of supernatant and put it in a 15ml centrifuge tube, then centrifuge at 16000g for 10min at 4℃, take out about 4ml of supernatant, and put it in a 1.5ml centrifuge tube. Store frozen at -80°C. Plasma cfDNA was extracted using QIAamp @ ciculiatiq NucleicAcid KIT, and finally eluted with 50ul EB buffer. The sample name and extracted cfDNA concentration and quality information are shown in Table 4:

[0031]

[0032] Synthesize adapters with random labels as shown in the above table 1, wherein, N represents any one of the four bases of ATCG, and every two random bases are separated by an A or T or C or G base, A total...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6806
CPCC12Q1/6806C12Q1/6869C12Q2535/122C12Q2525/191C12Q2531/113
Inventor 刘青青谢文龙曾缘欢陈荣山李宇温恒斌肖辛野姚迅李奇渊李晓哲
Owner 厦门基源医疗科技有限公司
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