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A reaction system for double-end sequencing, its composition kit and application

A paired-end sequencing and reaction system technology, applied in the field of DNA sequencing, can solve the problems of low sequencing signal, poor sequencing quality, and low complementary strand sequencing signal, and achieve the effects of reducing Runon value, improving efficiency, and improving accuracy

Active Publication Date: 2022-01-18
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing method is to use the conventional MDA method for complementary strand generation followed by sequencing, which results in low sequencing signal and poor sequencing quality
[0005] The current DNB complementary chain generation method has two deficiencies: (1) the complementary chain sequencing signal is low; (2) the complementary chain sequencing index Lag&Runon (Lag refers to the lagging of base synthesis during sequencing, and Runon refers to the advanced base synthesis during sequencing) is obvious Higher, affecting the quality of sequencing, how to provide the sequencing signal of the complementary strand, how to reduce the sequencing index Lag&Runon has become an urgent problem to be solved

Method used

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  • A reaction system for double-end sequencing, its composition kit and application
  • A reaction system for double-end sequencing, its composition kit and application
  • A reaction system for double-end sequencing, its composition kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Take the BGISEQ500 platform as an example to demonstrate the effect of adding different combinations of PCR enhancers in the reaction system

[0065]The method for paired-end sequencing comprises the steps of:

[0066] (1) Referring to the operating instructions of the BGISEQ-500 sequencer, load the DNA nanospheres on the chip, use the single-end sequencing primer (SEQ ID NO.1) and supporting reagents (BGISEQ-500RS high-throughput kit PE50 V2.0 ) sequencing the 5' end of the DNA nanosphere;

[0067] Specifically, the nucleotide sequence of the sequencing primer SEQ ID NO.1 is: 5'-GCTCACATCAGGCCATTAGGCTACGAGACTT-3';

[0068] (2) After the 5' end sequencing is completed, use the excision reagent (CPAS regeneration reagent (Regeneration Reagent), from BGISEQ-500RS high-throughput kit PE50 V2.0) to excise the fluorescence and blocking base of the last base group, and then use the elution reagent (from BGISEQ-500RS high-throughput kit PE50 V2.0) to remove the ex...

Embodiment 2

[0107] Example 2: Taking the BGISEQ500 platform as an example, it is shown that adding 1 mM CTAB to the complementary strand sequencing primer solution can reduce the sequencing Lag&Runon value.

[0108] The method for paired-end sequencing comprises the steps of:

[0109] (1) Referring to the operation manual of the BGISEQ500 sequencer, load the DNA nanospheres on the chip, and use the single-end sequencing primer (SEQ ID NO.1) and supporting reagents (BGISEQ-500RS high-throughput kit PE50 V2.0) The 5' end of the DNA nanosphere is sequenced;

[0110] Specifically, the nucleotide sequence of the sequencing primer SEQ ID NO.1 is: 5'-GCTCACATCAGGCCATTAGGCTACGAGACTT-3';

[0111] (2) After the 5' end sequencing is completed, use the excision reagent (CPAS regeneration reagent (Regeneration Reagent), from BGISEQ-500RS high-throughput kit PE50 V2.0) to excise the fluorescence and blocking base of the last base group, and then use the elution reagent (from BGISEQ-500RS high-through...

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Abstract

The invention provides a reaction system for paired-end sequencing, a kit for its composition and applications, and specifically relates to a reaction system for improving the quality of paired-end sequencing, a kit for its composition, and a method for improving the quality of paired-end sequencing. The reaction system includes 1×Phi29 buffer, dNTP, Phi29 polymerase and PCR enhancer. In the present invention, a combined PCR enhancer is added to the reaction system for generating complementary chains, through mutual promotion among glycerin, DMSO, Triton X‑100, trehalose and betaine, a synergistic effect is exerted, and the complementary chains can be significantly improved. The efficiency of synthesis improves the accuracy of sequencing, and the average signal value of sequencing increases by 1.1 times; at the same time, the complementary strand generated by CTAB treatment limits the exo-cutting activity of Phi29 enzyme and significantly reduces the Lag&Runon value.

Description

technical field [0001] The present invention relates to the technical field of DNA sequencing, and relates to a reaction system for paired-end sequencing, its composition kit and application, in particular, to a reaction system for improving the quality of pair-end sequencing, its composition kit and its application. Methods for paired-end sequencing quality. Background technique [0002] Multiple displacement amplification (MDA) is currently recognized as the best single-cell genome amplification technology, which can uniformly amplify the whole genome with high fidelity, amplify fragments of 10-100kb size, and provide a large number of uniform and complete whole genome sequence. MDA technology is widely used in whole-genome amplification, and it is currently the whole-genome amplification method with the widest coverage of the entire genome and the smallest amplification bias at each site. Currently, this method is also used in the field of high-throughput sequencing. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/6806C12N15/10
CPCC12Q1/6806C12Q1/6869C12Q2527/125C12Q2535/122C12Q2521/101C12Q2531/125
Inventor 王卉杨晋陈奥徐崇钧章文蔚
Owner MGI TECH CO LTD