Double-platform combined peripheral blood cfDNA base mutation and methylation detection method

Abstract

The invention provides a double-platform combined peripheral blood cfDNA base mutation and methylation detection method, which solves the problem of how to obtain effective nucleic acid mutation information and modification information under the practical condition that the initial quantity of a sample is insufficient by using the design of a universal Y-shaped joint adaptive to an Illumina Nanner next-generation sequencing platform and a nanopore sequencing platform. The nucleic acid short fragment library connected with the Y-type connector is directly sequenced by using a second-generation platform, so that high-accuracy mutation information of fragmented nucleic acid can be obtained; after the nucleic acid short fragment library connected with the Y-shaped connector is subjected to enzyme digestion treatment, the fragmented nucleic acid is connected into a long fragment (gt; through the nanopore, the utilization rate of the nanopore, the effective read length and the total data volume are greatly improved, and meanwhile, the modification information of the fragmented nucleic acid is obtained. After the data is output, the multi-sample data can be split by utilizing the label design on the joint, so that the detection flux can be increased in order of magnitude, and the detection cost of a single sample is reduced.

Description

technical field [0001] The invention relates to the technical field of DNA methylation detection, in particular to a method for detecting base mutations and methylation of cfDNA in peripheral blood combined with dual platforms. Background technique [0002] Circulating tumor DNA (ctDNA) is a free gene fragment released into the peripheral blood circulation system by apoptosis or rupture of solid tumor cells. Therefore, ctDNA carries the same genetic defects as its source tumor DNA, such as point mutations, rearrangements, amplifications, microsatellite changes, epigenetic modifications, etc., and can be used as an alternative source of tumor DNA. Recent studies have shown that ctDNA serves as a diagnostic, prognostic, and monitoring marker for cancer patients by enabling non-invasive "liquid biopsies," blood tests that allow molecular detection of solid malignancies. [0003] Gene mutation refers to a series of heritable mutations in genomic DNA molecules. It has attracted ...

AI Technical Summary

Problems solved by technology

The bisulfite conversion sequencing method converts unmethylated cytosine into uracil and then detects it by next-generation sequencing, but the disadvantage is that the conversion efficiency is limited and short-read sequencing cannot accurately identify repetitive regions of the genome; immunoprecipitation sequencing Able to detect DNA or RNA methylation modifications, but not single-base resolution

[0008] In the application of nanopore sequencing technology, the speed of DNA single strand passing through the nanopore (~500bp / s) will have a great impact on the accuracy and effectiveness of sequencing, and the sequencing quality of short fragments (<500bp) is low or cannot be read effectively. Therefore, directly constructing and sequencing the cfDNA library obtained from peripheral blood will lead to low data quality or sequencing failure, which will directly lead to the inability of this platform to be put into use in the field of cfDNA sequencing

Researchers have found that short fragments of nucleic acids can be connected to long chains for nanopore sequencing, but the single-base accuracy of the connected fragments is low, only about 90%

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