Method for introducing molecular tag in library construction suitable for Illumina next-generation sequencing platform, linker sequence and application thereof

A linker sequence and library construction technology, which is applied in the field of high-throughput sequencing library construction, can solve problems such as errors, time-consuming, and high quality requirements for initial DNA.

Pending Publication Date: 2019-12-31
远辰生物科技(苏州)有限公司
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Problems solved by technology

The traditional library construction method is cumbersome and time-consuming. During the PCR amplification process, a large number of copy duplications will be randomly generated. When calculating the mutation f...

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  • Method for introducing molecular tag in library construction suitable for Illumina next-generation sequencing platform, linker sequence and application thereof
  • Method for introducing molecular tag in library construction suitable for Illumina next-generation sequencing platform, linker sequence and application thereof
  • Method for introducing molecular tag in library construction suitable for Illumina next-generation sequencing platform, linker sequence and application thereof

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Embodiment Construction

[0083] The present invention will be described in further detail below through specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0084] Preparation of Tn5 transposase complex

[0085] Use any one of the sequences shown in SEQ ID No.1-SEQ ID No.64 and the sequence shown in SEQ ID No.129 to assemble branched chain A in equimolar numbers. The assembly process is carried out on a PCR machine. The program is as follows : Incubate at 95°C for 5 minutes, then cool down to 60°C at a rate of 0.1°C per second and incubate for 15 minutes, then cool down to 25°C at a rate of 0.1°C per second.

[0086] Use the sequence shown in SEQ ID No.65-SEQ ID No.128 and the sequence shown in SEQ ID No.129 to assemble branched chain B in equimolar numbers. The assembly process is carried out on a PCR machine, and the program is as follows: Incubation at 95°C 5min, then cool...

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Abstract

The invention provides a method for introducing a molecular tag in library construction suitable for an Illumina next-generation sequencing platform, a linker sequence and a design method, a method for constructing a library by using the linker sequence, and a kit containing the linker sequence. Wherein the linker sequence comprises 5'-P-E-F-Q-3', P is a sequencing platform pairing sequence, E isa random base sequence composed of the same base, F is a given base sequence, and Q is a Tn5 transposase terminal recognition sequence. The linker sequence and the library construction method providedby the invention can successfully construct the library for DNA samples as low as 50 pg, and are suitable for preparing DNA library construction kits and disease diagnosis kits.

Description

technical field [0001] The present invention relates to a high-throughput sequencing library construction method, in particular to a library suitable for the Illumina next-generation sequencing platform, especially a method for introducing molecular tags in library construction, a linker sequence, a linker sequence design method, and applications. Background technique [0002] Next-generation sequencing is also called NGS sequencing or high-throughput sequencing. As the name suggests, high-throughput sequencing has a high throughput of sequencing reactions. We can get 1Gb to 3Tb of sequencing data in one sequencing reaction. The human genome includes 20,000 genes encoded by it. The remaining gene sequences can be obtained at one time. The accuracy of high-throughput sequencing is also very high, with a single-base accuracy exceeding 99.9%. The high-throughput sequencing platform has achieved breakthroughs from scientific research to clinical application, and is widely used ...

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Application Information

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IPC IPC(8): C12Q1/6876C12Q1/6806
CPCC12Q1/6876C12Q1/6806C12Q2525/191C12Q2531/113
Inventor 陈鑫泽
Owner 远辰生物科技(苏州)有限公司
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