Joint enclosing sequence, library construction kit, and construction method of sequencing library

A technology of adapter blocking sequences and blocking reagents, which is applied in the field of sequencing library construction, can solve the problem of low capture efficiency of target fragments, achieve the effects of reducing non-target region capture, increasing binding efficiency, and improving capture efficiency

Inactive Publication Date: 2018-08-28
天津诺禾致源生物信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to provide an adapter blocking sequence, a library construction kit and a method for constructing a sequencing library, so as to solve the problem of low capture efficiency of target fragments in the sequencing library in the prior art

Method used

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  • Joint enclosing sequence, library construction kit, and construction method of sequencing library
  • Joint enclosing sequence, library construction kit, and construction method of sequencing library
  • Joint enclosing sequence, library construction kit, and construction method of sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The reagents used in Example 1 are shown in Table 1, wherein the hybrid capture reagent is the RNA hybrid capture system of Agilent.

[0042] Table 1:

[0043]

[0044] 1. Sample extraction

[0045] Tissue samples were extracted using a paraffin-embedded tissue DNA extraction kit (article number Dp331) from Tiangen Company, and the extracted DNA was stored at -20°C for later use. See Table 2 for sample names and extraction volumes.

[0046] Table 2:

[0047]

[0048] 2. Fragmentation of DNA.

[0049] 2.1---DNA interruption

[0050] Take 6 copies of the sample (520ng / part) and use a Covaris disruption instrument (instrument model S220), and perform DNA fragmentation according to the parameters set in Table 3 below. The main peak of the fragmented product was at 200-350bp, and 20ng was taken for electrophoresis detection.

[0051] table 3:

[0052] Maximum incident power

work factor

Energy Transfer Points

processing time

450W

30...

Embodiment 2

[0202] The specific reagents used in Example 2 are shown in Table 18, wherein the hybridization capture reagent is the DNA hybridization capture system of IDT Company.

[0203] Table 18:

[0204]

[0205] 1. Sample extraction

[0206] Cell line samples were extracted using Tiangen’s Magnetic Bead Method Blood Genomic DNA Extraction Kit (Product No. DP329-02), and the extracted DNA was stored at -20°C for later use. See Table 19 for sample names and extraction volumes.

[0207] Table 19:

[0208]

[0209] 2. Fragmentation of DNA.

[0210] 2.1---DNA interruption

[0211] Take 6 copies of the sample (120ng / part) and use a Covaris disruption instrument (instrument model S220), and perform DNA fragmentation according to the parameters set in Table 20 below. The main peak of the fragmented product was at 200-350bp, and 20ng was taken for electrophoresis detection.

[0212] Table 20:

[0213] Maximum incident power

work factor

Energy Transfer Points

...

Embodiment 1

[0369] The result detection of embodiment one and embodiment two:

[0370] Use bwa software to compare the sequencing results of Example 1 and Example 2 with the reference genome. The specific quality control information is shown in Table 35 below, and the capture efficiency of libraries using different blocking reagents is shown in Table 36 below.

[0371] Table 35:

[0372]

[0373]

[0374] Table 36:

[0375]

[0376]

[0377] Attachment: Probe capture ratio = total bases covering the target region / total bases covering the whole genome * 100%.

[0378] From the above description, it can be seen that the above-mentioned embodiments of the present invention have achieved the following technical effects: compared with the unmodified blocking sequence, the present invention adds modification (especially reverse dT modification) at the 3' end The adapter blocking sequence improves the binding efficiency with the adapter, improves the blocking efficiency, reduces th...

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PUM

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Abstract

The invention provides a joint enclosing sequence, a library construction kit, and a construction method of a sequencing library. The joint enclosing sequence comprises a P5 joint enclosing sequence containing a sequence represented by SEQ ID NO:1 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC, and the 3' terminal of the sequence represented by SEQ ID NO:1 is modified through one modification selected from reverse dT modification, dividing wall modification, dideoxycytidylic acid modification, and phosphorylation modification; the 3' terminal of the joint enclosing sequence is provided with enclosing modification, so that enclosing sequence combination efficiency is increased, non-target region capturing caused by joint connection is reduced, capturing efficiency is increased, and influences of the enclosing sequence on subsequent PCR process are reduced.

Description

technical field [0001] The invention relates to the field of sequencing library construction, in particular to a linker closure sequence, a library construction kit and a method for constructing a sequencing library. Background technique [0002] The general process of using Illumina TrueSeq adapters for library construction is as follows: first, fragment the DNA into 300-400bp, perform end repair on the fragmented DNA, and add A base tails, then connect the library with Illumina TrueSeq adapters, and finally use The known universal P5 and P7 sequences at both ends of the adapter are used as primers for PCR amplification and enrichment, and the constructed library can be used for hybridization capture. [0003] Before the hybrid capture process, the library needs to be blocked, on the one hand, to block the repeat sequence in the insert fragment, and on the other hand, to block the library adapter sequence. The closed library captures the target region with a capture probe,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C40B50/06C12N15/11
CPCC12Q1/6806C12Q2600/156C40B50/06C12Q2565/519C12Q2525/191C12Q2535/122
Inventor 李萍梁永单光宇高连菊臧晚春
Owner 天津诺禾致源生物信息科技有限公司
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