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WO2018129644A1 - Procédé, système de réaction et kit pour améliorer la qualité de séquençage par polymérisation d'acide nucléique - Google Patents

Procédé, système de réaction et kit pour améliorer la qualité de séquençage par polymérisation d'acide nucléique Download PDF

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Publication number
WO2018129644A1
WO2018129644A1 PCT/CN2017/070711 CN2017070711W WO2018129644A1 WO 2018129644 A1 WO2018129644 A1 WO 2018129644A1 CN 2017070711 W CN2017070711 W CN 2017070711W WO 2018129644 A1 WO2018129644 A1 WO 2018129644A1
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WIPO (PCT)
Prior art keywords
nucleotide
sequencing
nucleic acid
modification
type
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Ceased
Application number
PCT/CN2017/070711
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English (en)
Chinese (zh)
Inventor
李计广
马可心
邱敏
陈奥
徐崇钧
章文蔚
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Publication date
Application filed by MGI Tech Co Ltd filed Critical MGI Tech Co Ltd
Priority to CN202210627105.4A priority Critical patent/CN114958998A/zh
Priority to CN202210628687.8A priority patent/CN115141880A/zh
Priority to PCT/CN2017/070711 priority patent/WO2018129644A1/fr
Priority to CN201780068192.1A priority patent/CN109937259B/zh
Publication of WO2018129644A1 publication Critical patent/WO2018129644A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present application relates to the field of nucleic acid sequencing, and in particular to a method, a reaction system and a kit for improving the quality of nucleic acid polymerization sequencing.
  • Nucleic acid polymerization sequencing ie, side-synthesis sequencing (abbreviated SBS) refers to the ability to add fluorescently labeled nucleotides during the SBS process (Metzker et. al., Genome Res 15(12): 1767-1776 (2005)). Helps identify template DNA bases (Prober et. al., Science 238:336-341 (1987)) to give DNA sequence information. In recent years, compared with other sequencing methods, polymerization sequencing has been favored for its high throughput and low price.
  • SBS side-synthesis sequencing
  • a reversible termination and a base with a fluorescent signal are synthesized by a DNA polymerase; specifically, a nucleotide having a modification at the 3' sugar hydroxyl group is used, thereby blocking the addition of other nucleotides, Fluorescent signals to distinguish between 4 different bases. After removal of the blocking and fluorophores, the naturally free 3' hydroxyl group is restored for addition to the next nucleotide and the fluorescent signal is removed to facilitate synthesis and detection of the next base.
  • the synthesis efficiency is very high, and in the case of incomplete synthesis in multiple copies, signal confusion will occur, affecting the read length and accuracy of sequencing.
  • Nucleotides with reversible blocking and fluorophores are required to be added in sequencing while synthesis. Due to the increased modification, the molecules of the nucleotides are larger, the molecular structure is special, and the nucleotides are not modified. In contrast, DNA polymerases are less efficient at identifying and synthesizing such modified dNTPs, limiting the read length and accuracy of sequencing.
  • the present application discloses a method for improving the quality of nucleic acid polymerization sequencing comprising the addition of a second type of nucleotide to the reaction sequence of the polymerization sequence, the 3' sugar hydroxyl group of the second type of nucleotide having a blocking modification but no fluorescent modification.
  • dNTPs having only blocking modification that is, a second type of nucleotide
  • the effect of blocking modified dNTPs on DNA polymerase recognition and synthesis is relatively small. Therefore, the addition of the second type of nucleotides can complement the incomplete reaction, increase the efficiency of the synthesis reaction, and reduce the risk of insufficient reaction; And because of the second category Nucleotides are not fluorescently modified, which reduces signal interference caused by unclear fluorescence refraction or unclean fluorescence of the excised cells, which improves the efficiency of excision and reduces the sequencing error rate; thereby improving the quality of sequencing.
  • the second type of nucleotide is used in the same amount as the first type of nucleotide in the reaction solution, and the first type of nucleotide is a 3' sugar hydroxyl group having both a blocking modification and a fluorescent modification.
  • the first nucleotide in the present application is a nucleotide which is normally added in the polymerization sequencing.
  • the second nucleotide and the first nucleotide are added in equal amounts.
  • the amount of the second type of nucleotide can be adjusted according to requirements, but as long as the second type of nucleotide is added, the synthesis reaction efficiency can be improved to a certain extent. The effect of sequencing error rate.
  • Another aspect of the present application discloses the use of the method of the present application in nucleic acid sequencing.
  • the method of the present application is applicable to the sequencing of nucleic acid sequences of the human genome and other animal, plant and microbial species; the main applications include sequencing of WES, WGS, RNA, DNA, etc.; in one embodiment of the present application, the method of the present application is applied to the large intestine DNA sequencing of Bacillus.
  • a further aspect of the present application discloses a nucleic acid sequencing method, which comprises simultaneously adding two types of nucleotides to a reaction solution in a process of sequencing while synthesizing, the first nucleotide being a 3' sugar hydroxyl group and having a blocking effect.
  • Modified and fluorescently modified nucleotides the second type of nucleotide is a 3' sugar hydroxyl group having only a blocking modification but no fluorescent modification.
  • the method for improving the quality of nucleic acid polymerization sequencing of the present application can be applied to various sequencing platforms based on edge synthesis sequencing, and the application only needs to be added to the reaction solution without changing the sequencing conditions and parameters.
  • the second type of nucleotide can be.
  • Another aspect of the present application discloses a reaction system for improving the quality of nucleic acid sequencing, comprising a reaction solution for sequencing while synthesizing, wherein a second type of nucleotide is added to the reaction solution, and a third nucleotide of the second type is added.
  • the saccharide hydroxyl group has a blocking modification but no fluorescent modification.
  • the reaction solution comprises a reaction buffer, a DNA polymerase and a first type of nucleotide, and the first type of nucleotide is a 3' sugar hydroxyl group having both a blocking modification and a fluorescent modification.
  • a further aspect of the present application discloses a nucleic acid sequencing kit comprising the reaction system of the present application.
  • the key inventive idea of the present application is to add dNTPs having only blocking modification, that is, a second type of nucleotide, in the reaction solution which is synthesized while synthesizing, so as to improve the synthesis efficiency;
  • the present application further proposes a reaction system for improving the quality of nucleic acid sequencing, that is, a reaction solution prepared by sequencing while adding a second type of nucleotide.
  • a reaction system for improving the quality of nucleic acid sequencing that is, a reaction solution prepared by sequencing while adding a second type of nucleotide.
  • the reagent system of the present application can also be made into a nucleic acid sequencing kit.
  • the method for improving the quality of nucleic acid polymerization sequencing of the present application adds dNTPs having only blocking modification to the reaction liquid, and since such dNTPs have no fluorescent modification, it is more conducive to DNA polymerase recognition and synthesis, and can supplement the incomplete reaction portion. Increasing the efficiency of the synthesis reaction and reducing the risk of insufficient reaction; at the same time, because there is no fluorescence modification, the signal interference caused by the unclear fluorescence removal or the uncleaned fluorescence of the resection is reduced, and the resection efficiency is improved and the reduction is improved. Sequencing error rate.
  • FIG. 1 is a schematic structural view of a second type of nucleotide in the embodiment of the present application.
  • FIG. 2 is a schematic structural view of a first type of nucleotide in the embodiment of the present application.
  • Figure 5 is a graph showing the variation in sequencing error rate for each cycle in the examples of the present application.
  • the present application proposes to add dNTPs having only blocking modification on the basis of the existing polymerization sequencing reaction solution;
  • the second type of nucleotide can be used to supplement the incomplete part, which guarantees the synthesis efficiency and reduces the risk of insufficient reaction;
  • the fluorescence excision is also reduced. Unclean, or signal interference caused by unclear fluorescent elution, improves the efficiency of resection and reduces the sequencing error rate.
  • the nucleic acid sequencing method of this example was performed on a BGISEQ-500 sequencing platform, and the sequenced sample was an E. coli standard library.
  • the specific plan is as follows:
  • the dNTP 1 mixture is prepared, that is, the first nucleotide; the first nucleotide of this example, as shown in FIG. 2, has both a fluorescent dye and a blocking group on the nucleotide.
  • A, T, G, and C represent adenine nucleotides, thymidine nucleotides, guanine nucleotides, and cytosine nucleotides, respectively.
  • dNTP 1 mixture Final concentration (nmol/L) dCTP_1 100 dGTP_1 100 dATP_1 100 dTTP_1 100
  • dCTP_1 refers to cytosine nucleotides with both blocking and fluorescent modifications
  • dGTP_1 refers to guanine nucleotides with both blocking and fluorescent modifications
  • dATP_1 refers to both blocking and fluorescent modifications.
  • the adenine nucleotide, dTTP_1 refers to a thymidine nucleotide having both blocking and fluorescent modifications.
  • the dNTP 2 mixture is configured, that is, the second type of nucleotide; the second nucleotide of this example, as shown in Figure 1, has only a blocking group on the nucleotide, and no fluorescence.
  • a dye wherein A, T, G, and C represent adenine nucleotides, thymidine nucleotides, guanine nucleotides, and cytosine nucleotides, respectively.
  • dNTP 2 mixture Final concentration (nmol/L) dCTP_2 100 dGTP_2 100 dATP_2 100 dTTP_2 100
  • dCTP_2 refers to a cytosine nucleotide having only a blocking modification
  • dGTP_2 refers to a guanine nucleotide having only a blocking modification
  • dATP_2 refers to an adenine nucleotide having only a blocking modification
  • dTTP_2 is Refers to a thymidine nucleotide that only has a blocking modification.
  • SE50 sequencing was performed using the BGISEQ-500 platform, the experimental group with the synthetic reagent 1 was added as the control group, and the experimental group with the synthetic reagent 2 was added as the experimental group, and the fluorescence signal curves of each cycle in the two groups were statistically analyzed. Sequencing quality change curve for each cycle and sequencing error rate curve for each cycle.
  • the sequencing results are shown in Figures 3 to 5.
  • the fluorescence signal change curve of each cycle is shown in Fig. 3.
  • the abscissa is the sequencing read length
  • the ordinate is the sequencing signal
  • “ ⁇ ” is the curve of the experimental group
  • “ ⁇ ” is the curve of the control group
  • the sequencing quality variation curve of each cycle is shown in Fig. 4.
  • the abscissa is the sequencing read length
  • the ordinate is the sequencing quality value
  • “ ⁇ ” is the curve of the experimental group
  • “ ⁇ ” is the curve of the control group
  • the sequencing error rate curve for each cycle is shown in Figure 5.
  • the abscissa is the sequencing read length.
  • the ordinate is the sequencing error rate
  • “ ⁇ ” is the curve of the experimental group
  • “ ⁇ ” is the curve of the control group; the results show that with the increase of the sequencing cycle, the sequencing error rate begins to increase, and the error rate of the experimental group increases faster than the comparison.
  • the group should be slow, indicating that the experimental group has a lower error rate.

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  • Organic Chemistry (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé, un système de réaction et un kit pour améliorer la qualité de séquençage par polymérisation d'acide nucléique. Le procédé d'amélioration de la qualité de séquençage de polymérisation d'acide nucléique de la présente invention consiste à : ajouter un second nucléotide dans une solution de réaction de séquençage par polymérisation, un groupe hydroxyle du saccharide en 3' du second nucléotide étant doté d'une modification de blocage mais pas d'une modification de fluorescence.
PCT/CN2017/070711 2017-01-10 2017-01-10 Procédé, système de réaction et kit pour améliorer la qualité de séquençage par polymérisation d'acide nucléique Ceased WO2018129644A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202210627105.4A CN114958998A (zh) 2017-01-10 2017-01-10 一种提高核酸聚合测序质量的方法、反应体系和试剂盒
CN202210628687.8A CN115141880A (zh) 2017-01-10 2017-01-10 一种提高核酸聚合测序质量的方法、反应体系和试剂盒
PCT/CN2017/070711 WO2018129644A1 (fr) 2017-01-10 2017-01-10 Procédé, système de réaction et kit pour améliorer la qualité de séquençage par polymérisation d'acide nucléique
CN201780068192.1A CN109937259B (zh) 2017-01-10 2017-01-10 一种提高核酸聚合测序质量的方法、反应体系和试剂盒

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PCT/CN2017/070711 WO2018129644A1 (fr) 2017-01-10 2017-01-10 Procédé, système de réaction et kit pour améliorer la qualité de séquençage par polymérisation d'acide nucléique

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Cited By (1)

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WO2024016625A1 (fr) * 2022-07-21 2024-01-25 深圳赛陆医疗科技有限公司 Procédé de séquençage de nouvelle génération rapide

Families Citing this family (1)

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CN120344676A (zh) * 2022-12-16 2025-07-18 深圳华大智造科技股份有限公司 一种降低双端同步测序错误率的方法

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CN102030792A (zh) * 2009-09-29 2011-04-27 韩国科学技术研究院 3＇-ｏ-荧光修饰的核苷酸及其用途
CN103602719A (zh) * 2013-04-07 2014-02-26 北京迈基诺基因科技有限责任公司 一种基因测序方法

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US20120156728A1 (en) * 2010-12-17 2012-06-21 Life Technologies Corporation Clonal amplification of nucleic acid on solid surface with template walking
WO2014031163A1 (fr) * 2012-08-24 2014-02-27 Life Technologies Corporation Procédés, compositions, systèmes, appareils et nécessaires utilisables en vue du séquençage d'extrémités appariées d'acides nucléiques
CN103951724B (zh) * 2014-04-30 2017-02-15 南京普东兴生物科技有限公司 一种特殊修饰的核苷酸及其在高通量测序方面的应用
IL255445B (en) * 2015-07-30 2022-07-01 Illumina Inc Orthogonal deblocking of nucleotides
WO2017027783A1 (fr) * 2015-08-13 2017-02-16 Centrillion Technology Holdings Corporation Procédés de synchronisation de molécules d'acide nucléique
EP3356381A4 (fr) * 2015-09-28 2019-06-12 The Trustees of Columbia University in the City of New York Dérivés nucléotidiques et leurs méthodes d'utilisation
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CN102030792A (zh) * 2009-09-29 2011-04-27 韩国科学技术研究院 3＇-ｏ-荧光修饰的核苷酸及其用途
CN103602719A (zh) * 2013-04-07 2014-02-26 北京迈基诺基因科技有限责任公司 一种基因测序方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024016625A1 (fr) * 2022-07-21 2024-01-25 深圳赛陆医疗科技有限公司 Procédé de séquençage de nouvelle génération rapide

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CN109937259A (zh) 2019-06-25
CN115141880A (zh) 2022-10-04
CN109937259B (zh) 2023-01-13
CN114958998A (zh) 2022-08-30

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