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Method for constructing sequencing library of target sequence

A technology for sequencing libraries and target sequences, applied in the field of building sequencing libraries of target sequences, can solve problems such as high detection cost, lower target sequence ratio, lower capture efficiency, etc.

Active Publication Date: 2021-06-01
BERRY ONCOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, such methods have the following disadvantages: the total length of the adapters at both ends of the members of the complete whole genome sequencing library (for example, from figure 1 The length of P5 / P7 to UMI shown) is usually about 60-80 nucleotides, and library members carrying adapters of this length bind to each other (or "lap") to form complexes during capture , thereby significantly reducing the proportion of the target sequence, resulting in a lower capture efficiency
[0007] In order to solve the problem of reduced capture efficiency due to the overlap of adapters of library members in conventional target sequence sequencing methods, adapter blocking agents are usually added in the probe hybridization stage of the capture process, which is specially designed and modified, and can be combined with DNA to which linker sequences efficiently bind to inhibit linker lapping (e.g. figure 2 shown)
However, due to the special design and modification of the joint sealer and the high concentration required to achieve the blocking effect, the detection cost is quite high

Method used

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  • Method for constructing sequencing library of target sequence
  • Method for constructing sequencing library of target sequence
  • Method for constructing sequencing library of target sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0312] Example 1: The library construction (C) of the linker of the present invention without a blocking agent, and the IDT commercial long linker control plus sealing Library construction with blocking agent (K), IDT commercial adapter control plus blocking agent library construction (M), IDT commercial long adapter pair According to the library construction without blocking agent (J1), and IDT commercial linker control library construction without blocking agent (J2) Compare

[0313] Sample: commercialized tumor mutation standard diluted to a certain mutation ratio (Jingliang Gene, tumor SNV 5% gDNA standard, Cat. No. GWOGTM1003).

[0314] Targets: 400K, including 86 tumor-related genes (IDT synthesized 120nt 5-end biotin-modified probe pool).

[0315] Linker: (1) linker of the present invention (31 nucleotides) (Example C, see sequence listing for sequence); (2) commercialized long linker control (xGen Dual index with UMI Adapter) purchased from IDT (for comparison ...

Embodiment 2

[0321] Example 2: The linker of various lengths of the present invention does not add the library construction of blocking agent, the library construction of long adapter control without adding blocking agent, and the library construction (CK) of IDT commercial long adapter control plus blocking agent Comparison.

[0322] Sample: same as Example 1.

[0323] Target: same as Example 1.

[0324]Linkers: (1) linkers of various lengths of the present invention (16 nucleotides, 20 nucleotides, 24 nucleotides, 31 nucleotides, 34 nucleotides, 37 nucleotides, 40 nucleotides); (2) the long linker control (62 nucleotides) designed by the inventors themselves according to the Illumina platform; and (3) the commercialized long linker control (same as Example 1) purchased from IDT (CK). See the sequence listing for linker sequences.

[0325] The first round of amplification: For adapters of various lengths in the present invention, an intermediate library (or pre-library) is prepared. ...

Embodiment 3

[0330] Example 3: Verified in clinical application, the comparison between the library construction of the adapter of the present invention without blocking and the library construction of IDT commercial adapter plus blocking agent.

[0331] Samples: fresh clinical tumor tissue samples from co-constructed laboratories (12 cases)

[0332] Target: 39M whole exome, derived from IDT xGen Exome Research Panel v1.0, Cat. No. 1056115.

[0333] Linker: (1) the linker of the present invention (31 nucleotides); and (2) a commercially available long linker control purchased from IDT (same as Example 1). See the sequence listing for linker sequences.

[0334] The first round of amplification: for the adapter of the present invention, an intermediate library (or pre-library) is prepared. For the commercially available long linker controls purchased from IDT, what has been constructed is already a complete genome-wide library. See the sequence listing for the corresponding primer sequenc...

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Abstract

The present invention relates to high throughput nucleic acid sequencing, and more particularly, to a method for constructing a sequencing library of target sequences and a corresponding kit.

Description

[0001] This application is a divisional application of an invention patent application with an application date of November 29, 2019, an application number of 201911207127X, and an invention title of "Method for Constructing a Sequencing Library of a Target Sequence". technical field [0002] The present invention relates to high-throughput nucleic acid sequencing, and more specifically, a method for constructing a sequencing library of target sequences and a corresponding kit. Background technique [0003] By efficiently enriching the target sequence to construct a sequencing library for the target sequence, it can effectively reduce the cost of sequencing and increase the depth of sequencing. For applications that typically require high-depth sequencing, such as somatic mutation detection, target enrichment performance (e.g., capture efficiency) is a major factor in determining method sensitivity and specificity. [0004] Currently, the commonly used target sequence sequen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12Q1/6806C40B50/06C12N15/1093C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 王寅李林蔚柳焱孙福明王柯张媛媛茹兰兰
Owner BERRY ONCOLOGY CO LTD