Evaluation method of linker connection efficiency in construction of next-generation sequencing library

A technology of next-generation sequencing library and adapter ligation, which is applied in the field of evaluating the efficiency of adapter ligation in the construction of next-generation sequencing library, and can solve problems such as the inability to accurately obtain the specific percentage of connection

Inactive Publication Date: 2020-09-15
烟台海利士科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the influence of the recovery rate of nucleic acid in the purification process and the introduction of joints and residual joints, this method cannot accurately obtain the specific percentage of connection.

Method used

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  • Evaluation method of linker connection efficiency in construction of next-generation sequencing library
  • Evaluation method of linker connection efficiency in construction of next-generation sequencing library
  • Evaluation method of linker connection efficiency in construction of next-generation sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] This specific example provides a method for evaluating adapter ligation efficiency in the construction of a next-generation sequencing library, comprising the following steps:

[0035] (1) Obtain fragment A by PCR amplification, use fragment A as a template, and use L1 and L2 as adapters to construct a next-generation sequencing library. After the adapter ligation is completed, use primers corresponding to adapters L1 and L2 as primers to connect The completed product is used as a template for PCR amplification, and the amplified fragments containing adapter sequences at both ends account for more than 99.999%. The fragments containing adapter sequences at both ends are L1-A-L2, where L1 and L2 are adapters, A is a DNA fragment;

[0036] (2) The method for measuring nucleic acid concentration by qubit is used to detect the nucleic acid concentration of L1-A-L2 constructed in step (1), and convert the nucleic acid concentration of L1-A-L2 into copy number;

[0037] (3) ...

Embodiment 2

[0043] This specific example provides another method for evaluating adapter ligation efficiency in next-generation sequencing library construction, including the following steps:

[0044](1) Use the human genome as a template to carry out PCR amplification to obtain fragment A, the amplified upstream primer sequence is shown in SEQ ID NO: 1, and the downstream primer sequence is shown in SEQ ID NO: 2, using fragment A as a template and L1 , L2 is the adapter for next-generation sequencing library construction. After the adapter ligation is completed, the primers corresponding to the adapters L1 and L2 are used as primers, and the products after the adapter ligation are used as templates for PCR amplification. Amplify until both ends contain adapter sequences Fragments accounted for more than 99.999%, and the fragments containing adapter sequences at both ends are L1-A-L2, where L1 and L2 are adapters, and A is a DNA fragment;

[0045] (2) The method for measuring nucleic acid ...

Embodiment 3

[0049] This specific example provides a method for evaluating adapter ligation efficiency in the construction of a next-generation sequencing library, comprising the following steps:

[0050] (1) Artificially synthesize a fragment containing linker sequences at both ends, which is L1-A-L2, wherein L1 and L2 are linkers, and A is a DNA fragment;

[0051] (2) The method for measuring nucleic acid concentration by qubit is used to detect the nucleic acid concentration of L1-A-L2 constructed in step (1), and convert the nucleic acid concentration of L1-A-L2 into copy number;

[0052] (3) Using L1 and L2 as linkers to construct a library for fragment A, the fragment A is artificially synthesized, and after the library construction is completed, the product of the library construction is purified and recovered to obtain library a;

[0053] (4) Using L1-A-L2 as the standard, carry out serial gradient dilution on the standard L1-A-L2, and then use the primers corresponding to fragment...

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Abstract

The invention discloses an evaluation method of linker connection efficiency in construction of a next-generation sequencing library. The method comprises the following steps of constructing a fragment containing linker sequences at two ends, wherein the fragment is L1-A-L2, L1 and L2 are linkers, and A is a DNA fragment; detecting to obtain the copy number of the constructed L1-A-L2; carrying outlibrary construction on the A fragment by taking L1 and L2 as linkers, and purifying and recycling a library construction product after the library construction is completed to obtain a library a; performing absolute quantification on the library a by adopting a qPCR method with L1-A-L2 as a standard substance, and detecting to obtain the number n of molecules of a total A fragment in the librarya and the number m of molecules of L1-A-L2 in the library a, wherein the percentage of the number of molecules connected with the linkers at the two ends in the library a in the total number of molecules of the A fragment is the linking efficiency of the linkers in library construction. Through the method disclosed by the invention, the specific percentage of the linker connection in the construction of the next-generation sequencing library can be accurately obtained.

Description

technical field [0001] The invention relates to the technical field of next-generation sequencing library construction, in particular to a method for evaluating adapter ligation efficiency in next-generation sequencing library construction. Background technique [0002] With the development and innovation of biotechnology, the requirements of existing scientific research and medical applications are getting higher and higher, and the original Sanger generation sequencing technology can no longer meet their needs. Next-generation sequencing technology (NGS) has greater advantages for genome sequencing because of its low cost, high speed, and high throughput. Next-generation sequencing technology performs end repair (Illumina platform library plus A) on nucleic acid fragments such as fragmented DNA, small fragments of DNA (such as free DNA), and double-stranded cDNA, and connects the adapters required for sequencing (nuclear DNA) at both ends of the fragments. nucleotides) to...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6855
CPCC12Q1/6851C12Q1/6855C12Q2531/113C12Q2525/191C12Q2545/113C12Q2537/165
Inventor 曹学彬张燕军黄秀付亮剑
Owner 烟台海利士科技有限公司
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