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An improved method for making random index adapters for next-generation sequencing

A production method and second-generation sequencing technology, which are used in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve the problems of high sequencing error rate, link failure, and loss of sequencing library templates, and achieve a good process. Compatibility, reduced production costs, reduced template loss, and wasted sequencing data volumes

Active Publication Date: 2019-12-13
AMOY DIAGNOSTICS CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The clinical application of next-generation sequencing still faces many problems and challenges, one of which is the high error rate of sequencing
[0007] However, due to the production process of the random index adapter, the final adapter will have some blunt-end adapters with incomplete enzyme digestion. The existence of this part of the blunt-end adapter will lead to the loss of the sequencing library template and the waste of the amount of sequencing data ( figure 1 , figure 2 )
Although the primer oligo method prepared by biotin-labeled adapters can remove the blunt end adapters, this method will increase the preparation steps of the adapters, and due to improper operation during the purification process of the avidin magnetic beads, it is easy to cause the melting of the double-stranded adapters. causing partial failure of the joint

Method used

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  • An improved method for making random index adapters for next-generation sequencing
  • An improved method for making random index adapters for next-generation sequencing
  • An improved method for making random index adapters for next-generation sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Improved linker preparation (dSpacer modified linker)

[0041]Connector primer P5, connector primer P7-A, connector primer P7-B, connector primer P7-C four primers (connector primer P5 is SEQ ID NO: 1; connector primer P7-A is FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNNNN connected in SEQ IDNO: obtained at the 5' end of 02; linker primer P7-B is FFFFF (Spacer)EEEEEKKKKNNNNNNNNNNNNNN connected at the 5' end of SEQ IDNO:02; linker primer P7-C is FFFFF(Spacer)EEEEELLLLLLNNNNNNNNNNNNNN connected at the end of SEQ IDNO:02 Obtained at the 5' end; synthetic manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd.) diluted with ddH2O (or TE buffer) to 100uM;

[0042] The FFFFF (Spacer) sequence is TCTTCTTC-dSpacer-T-dSpacer-TTCTTCT

[0043] The spacer modification site is dSpacer, EEEEE is the restriction site, JJJJJ / KKKKK / LLLLL is the positioning tag sequence, and NNNNNNNNNNNN is the random molecular tag sequence.

[0044] At the same time, FFFFF / EEEEE / JJJJJ / KKKKK / LLLLL...

Embodiment 2

[0051] Example 2: Improved linker preparation (spacer18 modified linker)

[0052] Connector primer P5, connector primer P7-A, connector primer P7-B, connector primer P7-C four primers (connector primer P5 is SEQ ID NO: 1; connector primer P7-A is FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNNNN connected in SEQ IDNO: obtained at the 5' end of 02; linker primer P7-B is FFFFF (Spacer)EEEEEKKKKNNNNNNNNNNNNNN connected at the 5' end of SEQ IDNO:02; linker primer P7-C is FFFFF(Spacer)EEEEELLLLLLNNNNNNNNNNNNNN connected at the end of SEQ IDNO:02 Obtained at the 5' end; synthetic manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd.) diluted with ddH2O (or TE buffer) to 100uM;

[0053] Where FFFFF (Spacer) sequence is TCTTCTTCTTC (spacer18) TTCTTCT

[0054] The spacer modification site is spacer18, EEEEE is the restriction site, JJJJJ / KKKKK / LLLLL is the positioning tag sequence, and NNNNNNNNNNNN is the random molecular tag sequence.

[0055] At the same time, FFFFF / EEEEE / JJJJJ / KKKKK / LLLL...

Embodiment 3

[0062] Example 3: Improved linker preparation (C6spacer modified linker)

[0063] Connector primer P5, connector primer P7-A, connector primer P7-B, connector primer P7-C four primers (connector primer P5 is SEQ ID NO: 1; connector primer P7-A is FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNNNN connected in SEQ IDNO: obtained at the 5' end of 02; linker primer P7-B is FFFFF (Spacer)EEEEEKKKKNNNNNNNNNNNNNN connected at the 5' end of SEQ IDNO:02; linker primer P7-C is FFFFF(Spacer)EEEEELLLLLLNNNNNNNNNNNNNN connected at the end of SEQ IDNO:02 Obtained at the 5' end; synthetic manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd.) diluted with ddH2O (or TE buffer) to 100uM;

[0064] Where FFFFF (Spacer) sequence is TCTTCTTCT (C6spacer) TC (C6spacer) TTCTTCT

[0065] The spacer modification site is C6 spacer, EEEEE is the restriction site, JJJJJ / KKKKK / LLLLL is the positioning tag sequence, and NNNNNNNNNNNN is the random molecular tag sequence.

[0066] At the same time, FFFFF / EEEEE / JJJ...

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Abstract

The present invention discloses an improved second-generation sequencing random label linker production method, which is characterized in that a Spacer modification capable of preventing the extension of a polymerase is introduced into a linker primer P7 in linker production; in a linker extension step, the extension is terminated after the polymerase encounters the Spacer site of a P7 template chain, and a 5' protruding terminal segment is leave; in the subsequent enzyme digestion step, the protruding terminal including the Spacer site is removed with endonuclease so as to produce a complete linker; and the residual un-digested linker being subjected to the incomplete enzyme digestion retains the long 5' protruding terminal.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to a process optimization for improved random index adapters for next-generation sequencing. Background technique [0002] With the maturity of technology and the reduction of cost, next-generation sequencing is gradually replacing traditional first-generation sequencing and conventional PCR molecular detection methods in the fields of scientific research and clinical transformation due to its advantages in sequencing throughput. [0003] The clinical application of next-generation sequencing still faces many problems and challenges, one of which is the high error rate of sequencing. At present, the widely used next-generation sequencing platforms mainly include Illumina platform (HiSeq, MiSeq, NextSeq, etc.) and Life's Ion Torrent platform (PGM, Proton and derivative series), and the sequencing error rate of the two types of next-generation sequencing is still relat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12N15/11C12N15/10
CPCC12Q1/6806C12Q2531/113C12Q2525/191C12Q2521/301
Inventor 金保雷李旭超施伟杰葛会娟阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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