CtDNA library construction and sequencing data analysis methods for simultaneously detecting common mutations of various liver cancers

Abstract

The invention discloses ctDNA library construction and sequencing data analysis methods for simultaneously detecting common mutations of various liver cancers. The library construction method and thesequencing data analysis method have the following advantages: firstly, various mutation forms of liver cancers are simultaneously detected under the condition that capture is not needed; secondly, the methods are suitable for efficient capturing in an ultra-small target area; thirdly, the library can support 10 to 20 times of detection; fourthly, the DNA barcode is connected to an initial ctDNA molecule during the library construction process and the flow is analyzed by cooperating with a biological information analysis process to realize high-specificity detection of ctDNA low-frequency mutation; and fifthly, the library can be used for PCR hot spot detection and capture method sequencing at the same time, the false positive mutation can be filtered effectively by using the added DNA barcode to realize duplex-based high-specificity sequencing. The methods have the important clinical significance for early screening, disease tracking, curative effect evaluation, prognosis prediction and the like of liver cancers.

Description

technical field [0001] The invention relates to a ctDNA library construction and sequencing data analysis method for simultaneously detecting multiple common mutations of liver cancer. Background technique [0002] ctDNA (circulating tumor DNA), that is, circulating tumor DNA, refers to the tumor DNA that exists in blood, cerebrospinal fluid and other body fluids and frees outside the cells. ctDNA is usually mixed with free DNA from normal cells in the blood, which is called cfDNA (cellfree DNA increases spaces). By detecting ctDNA mutations, it can guide targeted drug use, treatment monitoring, early screening of cancer, and so on. ctDNA-based detection methods include 1) PCR-based hotspot mutation detection method, which usually detects one or several hotspot mutations or known mutations, but cannot detect complex mutations such as gene fusions, and cannot detect unknown mutations. 2) Capture / next-generation sequencing method: It can detect positional mutations in many g...

AI Technical Summary

Problems solved by technology

ctDNA-based detection methods include 1) PCR-based hotspot mutation detection method, usually detects one or several hotspot mutations or known mutations, cannot detect complex mutations such as gene fusion, and cannot detect unknown mutations

2) Capture / next-generation sequencing method: It can detect positional mutations in many genes, including complex mutations, but capture kits are generally expensive, complicated to operate, and time-consuming

In addition, in ctDNA-related clinical testing or research, it is often necessary to compare the advantages and disadvantages of multiple techniques, which requires several times the normal blood volume, which is usually unacceptable to patients.

2) Regardless of the PCR method or the capture method, the noise mutations generated during the amplification process will seriously interfere with the detection of low-frequency mutations in ctDNA, resulting in false positive results and misleading the diagnosis and treatment of patients

3) The ctDNA mutation content is low, and contamination is prone to occur during the operation, resulting in false positive results

There is currently no simple, low-cost, and reliable solution for the systematic detection of ctDNA mutations in liver cancer

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