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Nucleic acid linker for high-throughput sequencing and library construction method

A technology for sequencing library and construction method, applied in the field of high-throughput sequencing, can solve the problems of long time consumption, adapter dimer pollution, high cost, and achieve the effect of reducing adapter self-ligation, reducing adapter self-ligation, and reducing self-ligation.

Active Publication Date: 2020-04-21
BEIJING USCI MEDICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are 2 steps of purification in the above 6 steps, and the steps are cumbersome; multiple purifications are time-consuming and costly; and multi-step purifications inevitably increase the loss of DNA samples
However, if the purification steps are simplified, for example, if the purification step of step (4) is omitted and the PCR process of step (5) is directly carried out, obvious contamination such as adapter dimers will occur, which will affect the quality of the subsequent library

Method used

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  • Nucleic acid linker for high-throughput sequencing and library construction method
  • Nucleic acid linker for high-throughput sequencing and library construction method
  • Nucleic acid linker for high-throughput sequencing and library construction method

Examples

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Effect test

Embodiment 1

[0053] Example 1 Adapter for high-throughput sequencing (1)

[0054] This embodiment provides a linker for high-throughput sequencing, which consists of linker 1 and linker 2. Linker 1 is a double-stranded oligonucleotide of unequal length, and its long-chain sequence is shown in SEQ ID NO.1. The short-chain The sequence is shown in SEQ ID NO.2, the 5' end of the long chain is modified by phosphorylation, the 3' end of the long chain and the 3' end of the short chain are modified by dideoxy; the linker 2 is a single-stranded oligonucleotide, and the sequence is as follows As shown in SEQ ID NO.3, linker 2 contains a region complementary to the 5' end of the long chain of linker 1.

[0055] The above-mentioned high-throughput sequencing adapters can be used for the construction of sequencing libraries on the BGI sequencing platform.

Embodiment 2

[0056] Example 2 Adapter for high-throughput sequencing (2)

[0057] This embodiment provides a linker for high-throughput sequencing, which consists of linker 1 and linker 2. Linker 1 is a double-stranded oligonucleotide of unequal length, and its long-chain sequence is shown in SEQ ID NO.4. The short-chain The sequence is shown in SEQ ID NO.5, the 5' end of the long chain is modified by phosphorylation, the 3' end of the long chain and the 3' end of the short chain are modified by dideoxy; the linker 2 is a single-stranded oligonucleotide, and the sequence is as follows As shown in SEQ ID NO.6, linker 2 contains a region complementary to the 5' end of the long chain of linker 1.

[0058] The above-mentioned high-throughput sequencing adapters can be used for sequencing library construction on the Illumina sequencing platform.

Embodiment 3

[0059] The construction of embodiment 3 sequencing library

[0060] In this example, cfDNA is used as the sequencing sample (parallel experiment with 3 samples), and the adapter of Example 1 is used to construct the sequencing library. The flow chart is as follows figure 2 As shown, the specific method is as follows:

[0061] 1. End repair and addition of A: The input amount of cfDNA is 2ng, and the reaction system is shown in Table 1; the reaction program: 65°C, 20min.

[0062] Table 1 End repair and A reaction system

[0063] Reagent Volume (μl) cfDNA 15 Taq enzyme 1 10x reaction buffer 2 dNTPS 1 h 2 o

1

[0064] 2. Joint connection: connect joint 1 and joint 2 in two steps, as follows:

[0065] The end-repaired and A-added product obtained in step 1 was connected to Adapter 1, and the reaction system was shown in Table 2; reaction program: 20°C, 15min.

[0066] Table 2 Connection reaction system of adapter 1

[0067] ...

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Abstract

The invention relates to the technical field of high-throughput sequencing, in particular to a nucleic acid linker for high-throughput sequencing and a library construction method. The nucleic acid linker comprises a linker 1 and a linker 2. The linker 1 is double-stranded unequal-length oligonucleotide and comprises a long strand and a short strand, the 5' end of the long strand is phosphorylated, and the 3' end of the long strand and the 3' end of the short strand are modifications capable of blocking formation of a 3' phosphodiester bond. The linker 2 is a single-stranded oligonucleotide. The linker 2 comprises a complementary region to the 5' end of the long strand of the linker 1. According to the linker, linker self-connection can be effectively reduced, PCR amplification can be directly carried out without purification after linker connection, a constructed library has no obvious linker dimer pollution, the loss of DNA samples is effectively reduced, and the efficiency and quality of library construction are improved.

Description

technical field [0001] The invention relates to the technical field of high-throughput sequencing, in particular to a nucleic acid adapter for high-throughput sequencing and a library construction method. Background technique [0002] With the development of sequencing technology, genome sequencing has become a routine scientific research and clinical diagnosis technology. High-throughput sequencing technology (next-generation sequencing technology, NGS) realizes massively parallel sequencing on high-density chips, which has the characteristics of high data output and low unit data cost, which greatly promotes the practical application of sequencing technology. During the NGS sequencing process, complex library construction steps are required for the sample DNA. The DNA loss problem during the library construction process will affect the sequencing quality. Especially in the face of low input, how to effectively reduce the multi-step purification process during the library c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6806C12N15/10C40B50/06C40B80/00
CPCC12Q1/6806C12N15/1093C40B50/06C40B80/00C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 孙广欣杨飘石露王冬伍启熹王建伟刘倩唐宇
Owner BEIJING USCI MEDICAL LAB CO LTD
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