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Sequencing joint and preparation method and application thereof in ultra-low frequency mutation detection

A sequencing linker and ultra-low frequency technology, which is applied in biochemical equipment and methods, DNA preparation, microbial measurement/inspection, etc., can solve the problem of insufficient detection accuracy and achieve the effect of improving detection accuracy

Active Publication Date: 2016-08-17
BEIJING KEXUN BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The main purpose of the present invention is to provide a sequencing adapter, its preparation method and its application in ultra-low frequency variation detection, so as to solve the problem of insufficient detection accuracy in the prior art

Method used

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  • Sequencing joint and preparation method and application thereof in ultra-low frequency mutation detection
  • Sequencing joint and preparation method and application thereof in ultra-low frequency mutation detection
  • Sequencing joint and preparation method and application thereof in ultra-low frequency mutation detection

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preparation example Construction

[0036] In another typical embodiment of the present application, a method for preparing a sequencing linker is also provided, the preparation method comprising: designing two single-stranded sequences of the sequencing linker, each single-stranded sequence containing an error prompt sequence, and The error prompt sequence is located on the side of the single-stranded sequence close to the target fragment to be connected, and the error prompt sequence is a sequence with a known base sequence; two single-stranded sequences are synthesized respectively; the two synthetic single-stranded sequences are specifically identified Anneal to form double-stranded sequencing adapters.

[0037] The synthesis in the above preparation method refers to chemical synthesis, which is usually synthesized by a company that provides chemical synthesis services. Rather than biosynthesis, that is, it does not refer to synthesis by using one strand as a template and the other through replication and ex...

Embodiment 1

[0075] 2. Embodiment 1 joint (2) manufacture method

[0076] (1) Linker sequence synthesis

[0077] ERROR-top: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNT (SEQ ID NO: 5);

[0078] ERROR-bot: P*NNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC (SEQ ID NO: 6: ). Remarks: P* is phosphorylation modification

[0079] In Example 1, 100 kinds of error prompt sequences were specifically set for N parts, and the ERROR-bot tag sequence was reversely complementary to the corresponding ERROR-top error prompt sequence. Among them, Table 4 shows 100 kinds of ERROR-top sequences. ERROR-top and the corresponding ERROR-bot are synthesized separately during synthesis. Among them, among the 7 bases shown in bold, except that the T at the end is for connecting with the protruding A at the 3' end of the target fragment, the remaining 6 bases are error prompt sequences.

[0080] Table 4:

[0081]

[0082]

[0083]

[0084]

[0085] (2) Dilute the powder of each primer with nuclease-free wat...

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Abstract

The invention provides a sequencing joint and a preparation method and application thereof in ultra-low frequency mutation detection. The sequencing joint comprises a library amplification primer sequence, a target fragment amplification primer sequence and an error prompting sequence which are connected sequentially, the error prompting sequence is close to one side of a target fragment, the library amplification primer sequence is located on one side away from the target fragment, and the error prompting sequence is the sequence of a known base sequence. The error prompting sequence of the known base sequence is added on one side close to the target fragment, so that the error prompting sequence can add a specific foreign marker on each double-strand DNA template. Following sequencing data of the target fragment can be obtained conveniently, mutation introduced in the sequencing or library amplification step is screened or removed according to the fact whether the sequencing sequences have same error prompting sequences, then the sites with variation in the same positions of the two chains are determined to be real mutation, and the site with the mutation on one chain is identified as the amplification or sequencing error, so that the mutation detection efficiency is improved.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing library construction, in particular to a sequencing linker, its preparation method and its application in ultra-low frequency variation detection. Background technique [0002] With the gradual maturity of high-throughput sequencing technology, its application fields are becoming more and more extensive. It can not only realize the detection from a large number of samples to trace samples, but also realize the detection of high-frequency mutations to ultra-low-frequency mutations. However, due to the current most accurate high-throughput sequencing itself, the error rate of a single base is only 10 -3 ~10 -2 or so, plus the well-known inherent error rate of DNA polymerases (10 -7 ~10 -5 ), therefore, it is impossible to achieve a detection accuracy of 1% or even higher by the current conventional experimental library construction method, and this error makes it difficult for us to dete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10C40B40/06C40B50/06C40B80/00
CPCC12Q1/6811C12Q1/6869C12Q2600/156C12Q2600/166C40B40/06C40B50/06C40B80/00C12Q2525/191C12Q2563/179C12Q2535/122
Inventor 周晔刘倩唐宇徐寒黎李伟伟郭现超李箐
Owner BEIJING KEXUN BIOTECH CO LTD
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