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DNA second-generation sequencing library, and construction method and construction kit thereof

A technology of next-generation sequencing library and construction method, applied in the field of DNA next-generation sequencing library and its construction method, and construction kit, which can solve the problems of low DNA quantity and failure

Inactive Publication Date: 2019-11-22
BEIJING NOVOGENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide a DNA next-generation sequencing library and its construction method and construction kit to solve the technical problem in the prior art that the library construction of FFPE samples with severe degradation is easy to fail due to low DNA quantity

Method used

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  • DNA second-generation sequencing library, and construction method and construction kit thereof
  • DNA second-generation sequencing library, and construction method and construction kit thereof
  • DNA second-generation sequencing library, and construction method and construction kit thereof

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Embodiment 1

[0029] In this embodiment, the main work is divided into two parts, that is, the first part of the experiment part and the second part of the data analysis part. In the first part, the samples to be tested are those judged by FFPE to be grade C (the grade judgment method is as follows: grade A, with main band or uniform degradation and the degradation area is mainly concentrated above 5000 bp; grade B, without main band, uniform degradation And the degradation area is mainly concentrated between 1500-5000bp; C grade, no main band, uniform degradation and the degradation area is mainly concentrated between 500-1500bp; D grade, no main band, the degradation area is scattered at 250-1000bp or concentrated in 500bp or less).

[0030] In this embodiment, the main reagent supplies are commercially available, and the information is shown in Table 1 below:

[0031] Table 1

[0032]

[0033]

[0034] The operation steps are as follows:

[0035] One: DNA fragmentation (refer to...

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Abstract

The invention discloses a DNA second-generation sequencing library, and a construction method and a construction kit thereof, wherein the construction method includes the following steps: a segmentedoriginal template DNA is obtained from a DNA sample by a method of enzyme digestion, and then the DNA second-generation sequencing library is constructed from the enzyme digestion product directly based on a method of single-strand joint connection without purification of the enzyme digestion reaction solution. With application of the technical scheme, the NGS sequencing library of the FFPE sampleis constructed based on the method of combining segmenting based on enzyme digestion with single-strand library construction, wherein the method simply comprises that the FFPE DNA is segmented by themethod of enzyme digestion, then the reaction product does not need to be purified, and the sequencing library is constructed directly by the method of single-strand library construction; and the method can be used for library construction of the FFPE sample with low initial amount and serious sample degradation, and improves the success rate of library construction and the number of original template detection.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a DNA next-generation sequencing library and its construction method and construction kit. Background technique [0002] FFPE (formalin-fixed paraffin embedded) samples have the characteristics of low sample volume and serious DNA degradation in the samples, which make FFPE samples extremely easy to fail in library construction. In conventional double-strand library construction, damaged DNA will be lost during PCR amplification after adapter ligation, resulting in the loss of a large number of templates. For single-strand library construction, the double-stranded DNA is first denatured to form a single strand. During this process, the damaged DNA can also form a fragmented single-stranded DNA, and then the single-stranded linker is connected to minimize the loss of the original template. [0003] The current library construction, due to the limitation of the read length of ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806C40B50/06C40B40/06
CPCC12N15/1093C12Q1/6806C40B40/06C40B50/06C12Q2525/191C12Q2531/113
Inventor 刘杨杨李瑞强李雷姚文钧宋迎楠成岗
Owner BEIJING NOVOGENE TECH CO LTD