A method for constructing a single-stranded DNA next-generation sequencing library for cfDNA

Abstract

The invention discloses a construction method of single stranded DNA next generation sequencing library specific to cfDNA. The construction method includes steps of 1), cfDNA extraction; 2), dephosphorylation; 3), denaturation to be single stranded cfDNA; 4), intermolecular single stranded connection on 3' end single stranded DNA interface; 5), extension: the acquired single stranded connection product is a template, and the known sequence design primer of the 3' end interface is extended; 6), intermolecular double stranded connection between the acquired double stranded DNA product and a 5' end double stranded DNA interface; 7), PCR amplification: use the acquired complete double stranded DNA interface connection product as a template, and take the known sequences of the interfaces at 3' end and the 5' end as forward and reverse products, and perform PCR amplification; 8), PCR product purification by a paramagnetic particle method, and library recycling. The original cfDNA gathering efficiency is high, and the double stranded, the single stranded and damaged DNA can be effectively detected.

Description

technical field [0001] The invention relates to the field of construction of a second-generation gene sequencing library, in particular to a method for constructing a single-stranded DNA next-generation sequencing library for cfDNA. Background technique [0002] cfDNA (cell-free circulating DNA) generally refers to DNA fragments present in plasma, urine and other body fluids, composed of double-stranded DNA, single-stranded DNA and damaged DNA, which are used in tumor monitoring, non-invasive prenatal and organ transplantation, etc. There are a wide range of applications in the field. [0003] Most of the current cfDNA next-generation sequencing technologies use conventional double-stranded DNA library construction methods, which can only detect undamaged double-stranded DNA, and in order to remove adapter dimer contamination, it is generally necessary to use magnetic beads The step of fragment selection, which leads to the loss of information of some small fragments of dou...

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Problems solved by technology

The ligation efficiency of this enzyme is relatively low, requires a long time for ligation, and requires biotin-labeled magnetic beads. The cost is high, the steps are complicated, and the loss of template DNA is also large, resulting in low enrichment efficiency of the original DNA. [2]

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