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Short joint, double-index joint primer and double-index library building system of gene sequencer

A technology of linker primers and linkers, applied in the field of short linkers, can solve the problems of poor uniformity of amplification efficiency, low fragment conversion efficiency, and poor uniformity of capture efficiency among index combinations.

Inactive Publication Date: 2021-04-09
北京吉因加医学检验实验室有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of low fragment conversion efficiency, poor uniformity of amplification efficiency among index combinations, poor uniformity of capture efficiency, and unstable sequencing data yield in the double-ended index joint library construction system in the prior art.

Method used

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  • Short joint, double-index joint primer and double-index library building system of gene sequencer
  • Short joint, double-index joint primer and double-index library building system of gene sequencer
  • Short joint, double-index joint primer and double-index library building system of gene sequencer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 short connector

[0044] short connectors, such as figure 1 Shown: the linker oligonucleotide chain 1 includes index2 primer binding region, sense linker complementary region, sense molecular tag and 1 protruding base T from the 5' end to the 3' end, and the sense linker complementary region All or part of it coincides with the 3' end sequence of the index2 primer binding region, and its nucleotide sequence is as follows; the linker oligonucleotide chain 2 includes the linker oligonucleotide chain in turn from the 5' end to the 3' end The antisense molecular label that is reverse complementary to the sense molecular tag in 1, the antisense adapter complementary region that is reverse complementary to the sense adapter complementary region of adapter oligonucleotide chain 1, and the index1 primer binding region, and the antisense adapter complementary region is all Or partially coincide with the 5' end sequence of the index1 primer binding region, and its n...

Embodiment 2

[0059] Example 2 double index linker primer

[0060] Double index linker primers, including forward linker primers and reverse linker primers;

[0061] Forward linker primer, including GF primer sequence, index2 sequence and index2 primer binding region sequence sequentially from 5' end to 3' end;

[0062] Reverse linker primer, including GR primer sequence, index1 sequence, index1 primer binding region reverse complementary sequence from 5' end to 3' end;

[0063] Such as figure 1 Shown: the nucleotide sequence of the forward adapter primer is as follows; the nucleotide sequence of the reverse adapter primer is as follows; specifically as follows:

[0064] Forward linker primer 5'-3':

[0065] TCTCAGTACGTCAGCAGTT NNNNNNNNNNCAACTCCTTGGCTCA CA GAACGACATGGCTACGATCC ;

[0066] Reverse Adapter Primer 5'-3':

[0067] GGCATGGCGACCTTATTCAG NNNNNNNNNNTT ;

[0068] In the above sequence (refer to the sequence table SEQ ID NO.35-36), "NNNNNNNNNN" in the forward adapter p...

Embodiment 3

[0070] Example 3 Double index linker blocking sequence

[0071] Double index adapter blocking sequence: including forward adapter blocking sequence and reverse adapter blocking sequence;

[0072] The forward linker blocking sequence includes the reverse complementary sequence of the sense linker complementary region, the reverse complementary sequence of the index2 primer binding region, the reverse complementary sequence of the index2 sequence and the reverse complementary sequence of the GF primer sequence from the 5' end to the 3' end; All or part of the reverse complementary sequence of the sense linker complementary region coincides with the 5' end sequence of the reverse complementary sequence of the index2 primer binding region;

[0073] Reverse linker blocking sequence, including GR primer sequence, index1 sequence, index1 primer binding region reverse complementary sequence and antisense linker complementary region reverse complementary sequence from 5' end to 3' end;...

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Abstract

The invention provides a short joint, a double-index joint primer and a double-index library building system of a gene sequencer. The short joint comprises two partially complementary joint oligonucleotide chains: including a joint oligonucleotide chain 1 and a joint oligonucleotide chain 2, wherein the joint oligonucleotide chain 1 sequentially comprises an index 2 primer binding region, a positive-sense joint complementary region, a positive-sense molecular tag and a protruding base T from the 5' end to the 3 'end; and the joint oligonucleotide chain 2 sequentially comprises an antisense molecular tag, an antisense joint complementary region and an index1 primer binding region from the 5' end to the 3 'end. The fragment conversion efficiency, the amplification efficiency uniformity between index combinations, the capture efficiency uniformity and the data yield stability of the double-index library system constructed by using the short joint are remarkably superior to those of an existing double-index library system.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to a short linker of a gene sequencer, a double-index linker primer, and a double-index library construction system and their applications. Background technique [0002] The development of next-generation sequencing technology (NGS) has greatly improved the sequencing speed and throughput. The DNBSEQ-T7 launched by MGI has a single-day data output of up to 6Tb, which is 6000 times that of the Genome Analyzer ten years ago. Such a huge increase in throughput enables more samples to be sequenced simultaneously in the same Run. The current strategy to solve mixed sequencing of samples is to introduce sample-specific barcode sequences (index) on the DNA fragments of different samples. By identifying the index sequence information, the splitting of single-sample data is realized. In 2017, Illumina released a white paper that pointed out that in large-scale mixed sequen...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12N15/11C40B40/06C40B50/06C12Q1/6869
CPCC12Q1/6869C12Q1/6876C40B40/06C40B50/06
Inventor 杨玲楚玉星刘磊薛涛涛
Owner 北京吉因加医学检验实验室有限公司
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