Construction method of high-throughput sequencing library suitable for single-stranded DNA

A technology of sequencing library and construction method, applied in the field of high-throughput sequencing library construction, can solve problems such as inability to use single-stranded DNA, and achieve the effects of improving molecular utilization, avoiding molecular loss, and improving sensitivity

Pending Publication Date: 2020-03-24
ENVELOPE HEALTH BIOTECHNOLOGY CO LTD
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Problems solved by technology

This method uses high temperature to denature the double-stranded DNA into a single-strand before constructing the library. Not only can the double-stranded molecules in the

Method used

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  • Construction method of high-throughput sequencing library suitable for single-stranded DNA
  • Construction method of high-throughput sequencing library suitable for single-stranded DNA
  • Construction method of high-throughput sequencing library suitable for single-stranded DNA

Examples

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Embodiment 1

[0052] Example 1. High-throughput sequencing library construction method suitable for single-stranded DNA

[0053] The present invention is a method for constructing a high-throughput sequencing library. Using this method, single-stranded DNA can be used as a template for sequencing library construction. It is applicable to but not limited to sequencing platforms such as BGISEQ-500, MGISEQ, and Illumina. Applicable sample types include but are not limited to. Limited to fragmented DNA, cfDNA, and bisulfite-treated DNA, it can realize whole-genome sequencing, whole-genome methylation sequencing, and capture sequencing.

[0054] The high-throughput sequencing library construction method suitable for single-stranded DNA provided in this example, its technical core lies in a special linker (linker 1) with 6N (N is A or T or C or G) to make single-stranded DNA It is possible to connect the template with the adapter. The double-stranded linker with 6N can be randomly combined to th...

Embodiment 2

[0148] Example 2. Practical application of the method for constructing a high-throughput sequencing library suitable for single-stranded DNA of the present invention

[0149] cfDNA whole genome methylation sequencing (WGBS) was performed according to the technical process introduced in Example 1.

[0150] Preparation of library template: use MagPure Circulating DNA Maxi Kit to extract healthy human cfDNA, take 20ng of extracted cfDNA, and use EZ DNA Methylation-Gold TM Kit performs bisulfite conversion.

[0151] According to the relevant steps of Example 1, the WGBS library was constructed. After PCR purification, the library was quantified using Qubit. The total amount of the library was 378ng, and the library was 2100 such as image 3 shown. After the library construction is completed, the BGISEQ-500 platform is used for PE50 sequencing, and the expected WGBS sequencing data volume is 20G.

[0152] WGBS sequencing data analysis: BGISEQ-500 was used for sequencing, and the...

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Abstract

The invention discloses a construction method of a high-throughput sequencing library suitable for single-stranded DNA. The method comprises the following steps: linking a single-stranded template anda double-stranded linker 1 with degenerate bases (a special linker with degenerate bases enables the linking of the single-stranded template and the double-stranded linker to be possible, the double-stranded linker can be randomly combined to the single-stranded template to make a linker sequence be fully close to the template, and then the linking is completed by using T4 DNA ligase); extendingby using an extension primer to obtain a double-stranded product; linking the double-stranded product to a linker 2; and carrying out PCR amplification by taking the obtained linking product as a template to obtain the required library. According to the invention, the method can be used for constructing a high-throughput sequencing library, can further be used for constructing a single-stranded DNA library, can improve the utilization rate of single-stranded DNA in the construction of a high-throughput sequencing library, further achieves the purpose of low-initial-quantity library construction, and has key effect in early screening and prognosis monitoring application of tumors in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-throughput sequencing library construction method suitable for single-stranded DNA. Background technique [0002] Since the birth of the first high-throughput sequencing instrument in 2005, high-throughput sequencing methods have developed rapidly and brought forth new ones. With the continuous efforts of sequencing companies and researchers, technology updates and iterations have been completed (VAN DIJK EL, AUGER H, JASZCZYSZYN Y, et al. Ten years of next-generation sequencing technology [J]. Trends Genet, 2014, 30(9): 418-26.). The development of sequencing technology promotes the continuous progress of high-throughput sequencing, and the invention and improvement of library construction technology also play a pivotal role in the development of high-throughput sequencing. [0003] In the history of high-throughput sequencing technology development, researchers have develop...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6806C12Q1/6886
CPCC40B50/06C12Q1/6806C12Q1/6886C12Q2600/118C12Q2531/113C12Q2525/191
Inventor 李志隆汪宇盈叶明芝叶李莉刘继龙宋炎陈双双
Owner ENVELOPE HEALTH BIOTECHNOLOGY CO LTD
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