Sequencing method based on molecular tag and next-generation sequencing for reducing sequencing error, kit and application thereof

Abstract

The invention discloses a sequencing method based on a molecular tag and next-generation sequencing for reducing a sequencing error, a kit and an application thereof. The sequencing method comprises the following steps: S1, synthesizing 4 to 16 normal strands P5 and inverse strands P7 comprising different connectors of fixed specific sequence tags for each sample, annealing the P5 and P7, and forming a Y-shaped connector; S2, filling-in an end of a template DNA, and adding a basic group A on an end 3'; S3, connecting the Y-shaped connector to two ends of the processed template DNA; S4, recovering the template DNA connected with the connector, and gathering and amplifying the template DNA by utilizing a connector primer with the sample tag; S5, recovering a gathered and amplified product; and S6, performing the next-generation sequencing on the gathered and amplified product. By adopting the sequencing method, the mutation, and particularly the asymmetric mutation in a double-strand DNAoriginal template can be effectively identified, so that the amplification error introduced from the first gathering amplification can be effectively identified, and under an Illumina single-end anddual-end sample tag sequencing mode, the drift problem of the sample tag can be effectively solved by virtue of the fixed specific sequence of 4 to 16 unique connectors of a sample.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a sequencing method, kit and application thereof for reducing sequencing errors based on molecular tags and next-generation sequencing. Background technique [0002] Next-generation sequencing constructs a library, connects the adapter sequence to the target DNA template, and obtains more target DNA templates through enrichment amplification, and then performs deep sequencing on the constructed library on the next-generation sequencing platform. [0003] DNA polymerase is used in the library construction and platform sequencing of the above sequencing platforms. In order to ensure the accuracy of detection, high-fidelity DNA polymerase is used. The fidelity of this type of DNA polymerase is higher than 10 -9 , that is, every synthetic 10 9 1 base error occurs randomly. Therefore, the sequencing error of this technique is about 0.1%. [0004] Therefore, when the above technologies ...

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Problems solved by technology

[0007] The above techniques reduce sequencing errors by adding a random sequence of 9 bases at the P5 or P7 label position of the adapter, but since the positive and negative strands of the original template from the same double-stranded DNA have different labels, and the The combination of tags is uncertain, therefore, it is not possible to restore the positive and negative strands from the same double-stranded DNA original template.

Therefore, asymmetric mutations in the original template of double-stranded DNA cannot be identified

[0008] At the same time, during the first round of amplification of the enrichment amplification after ligation of the adapter with the label, there is a 10e-9 amplification error in the DNA polymerase, which will cause the amplification of the original template of the positive strand/negative strand. There are asymmetric mutations in the amplification product. When using

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