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WO2024138320A1 - Procédé de réduction de lieurs libres dans une banque de séquençage - Google Patents

Procédé de réduction de lieurs libres dans une banque de séquençage Download PDF

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Publication number
WO2024138320A1
WO2024138320A1 PCT/CN2022/142020 CN2022142020W WO2024138320A1 WO 2024138320 A1 WO2024138320 A1 WO 2024138320A1 CN 2022142020 W CN2022142020 W CN 2022142020W WO 2024138320 A1 WO2024138320 A1 WO 2024138320A1
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Prior art keywords
exonuclease
sequencing
sample
tested
optionally
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/142020
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English (en)
Chinese (zh)
Inventor
王欧
师虓
郭斐
陈俊毅
季州翔
章文蔚
董宇亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to PCT/CN2022/142020 priority Critical patent/WO2024138320A1/fr
Priority to CN202280102106.5A priority patent/CN120344725A/zh
Publication of WO2024138320A1 publication Critical patent/WO2024138320A1/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
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the prerequisite for the above research is how to use high-throughput parallel sequencing to obtain high-precision, complete nucleic acid sequences.
  • the sequencing accuracy of the current mainstream synthetic sequencing technology is relatively high, and the original accuracy of most sequenced bases can reach more than 99.9%.
  • its read length is short. Using these short read length sequences, whether it is resequencing or de novo assembly, it is difficult to restore the complete target nucleic acid sequence.
  • the Hi-Fi sequencing method based on circularization consensus sequencing launched by Pacbio in 2019 (Figure 1) can achieve an average sequencing accuracy of more than 99% by repeating the reading five times, but because it requires multiple repeated sequencing of the same molecule, the cost is relatively high.
  • Oxford Nanopore's latest PromethION 48 system can achieve a cost of $2-16 per Gb, which is gradually approaching the cost of synthetic sequencing, which is widely used in the market.
  • accuracy of this system is still insufficient compared with Pacbio's circularization consensus sequencing method, according to the latest data disclosed by Oxford Nanopore, its accuracy can reach 98.4%, which has been significantly improved compared with the early nanopore data.
  • nanopore sequencing libraries generally have longer inserts, ranging from a few thousand bases to a million bases, so they have fewer double-stranded ends than short insert libraries, and the efficiency of adapter connection is low. Since the nanopore sequencing adapters are generally coupled with the rate-controlling protein required for sequencing, it is impossible to use alcohol-containing cleaning reagents during magnetic bead or column purification after connection, making it difficult to remove the adapters in the purification step, resulting in the detection of non-target fragments during the sequencing process, resulting in a decrease in sequencing accuracy.
  • nuclease exonuclease can be added during the library construction process to remove residual connectors in the library.
  • no alcohol detergent is used, and only magnetic beads or column purification is used. This avoids the detection of non-target fragments during the sequencing process, improves the sequencing accuracy, increases the sequencing throughput, and obtains high-quality sequencing reads in a short time.
  • the method of an embodiment of the present invention by introducing a nuclease, the fragment to be tested that has not been connected at all, the single-stranded free connector that is not connected to the two ends of the inserted fragment to be tested, and the product of incomplete connector connection (referring to the product with only one end connected to the connector and the other end not connected to the connector) are degraded, thereby reducing the time occupied by the non-desired library in the sequencing channel time, thereby improving the sequencing throughput and increasing the sequencing accuracy.
  • the method further comprises subjecting the obtained digestion product to a detwisting treatment.
  • the unwinding process is performed under the action of a helicase.
  • the exonuclease recognizes double-stranded DNA and the exonuclease is a 5'->3' exonuclease, and the exonuclease includes at least one selected from T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, and Exonuclease VIII (truncated).
  • the nuclease is preferably T7 exonuclease.
  • the 3’->5’ exonuclease recognizes double-stranded DNA
  • the exonuclease includes at least one selected from Exonuclease III, Exonuclease IX, and Exonuclease X.
  • the exonuclease recognizes single-stranded DNA.
  • the method for establishing a sequencing library may further include performing anti-degradation modification on the 5' end and/or 3' end of the sample to be tested connected to the connector.
  • some exonucleases that can recognize the 5' end of single-stranded DNA and hydrolyze it (such as: T7 exonuclease, T5 exonuclease, Lambda exonuclease, Exonuclease VI, Exonuclease VIII (truncated)
  • the 5' end of the single strand such as the 5' end of the sequence to be tested connected to the Y-type connector, can be chemically modified in advance to resist degradation by nucleases.
  • the anti-degradation modification includes at least one selected from phosphate modification, 2'-OH modification (RNA base), 2'-F modification, LNA locked nucleotide modification and PNA peptide nucleic acid modification.
  • the digestion treatment is carried out at 37° C. and the ratio of the ligation product to T7 exonuclease is 44:1 for 4 to 6 minutes.
  • the non-Y-shaped linker has at least one of the following structures: complete complementary double strand, complementary double strand-non-complementary single strand-complementary double strand, 5' protruding single strand-complementary double strand, 3' protruding single strand-complementary double strand.
  • the method for establishing a sequencing library further comprises purifying the digestion product.
  • the purification process adopts Ampure XP magnetic beads purification.
  • the process before performing end-repair and A-addition treatment on the sample to be tested, the process further includes performing fragmentation treatment on the sample to be tested.
  • the present invention also proposes a sequencing library.
  • the sequencing library is a sequencing library of a nucleic acid sample obtained according to a method for establishing a sequencing library according to an embodiment of the present invention.
  • the inventors found that the sequencing library obtained by the method has significantly reduced the number of fragments to be tested that are not connected, free joints that are not connected to both ends of the inserted fragment to be tested, and products of incomplete joint connection, and is simple to operate, has significantly high sequencing accuracy, good repeatability, low cost, and high sequencing throughput.
  • FIG4 is a library construction process including an exonuclease step according to an embodiment of the present invention (sequencing direction is 3'->5');
  • FIG5 is a protein control result after purification according to an embodiment of the present invention.
  • iSp18 is an 18-atom-long hexaethylene glycol chain that is commonly used as a spacer in oligonucleotide chains.
  • Ad3 linker sequence was obtained by annealing chemically synthesized SEQ ID NO.1 and SEQ ID NO.2.
  • Buffer E 20 mM Tris-HCl pH 7.5, 100 mM NaCl.
  • step 4 Collect the Dda bacteria expressed in step 2, resuspend the bacteria with buffer A in step 3, break the bacteria with a cell disruptor, and then centrifuge to obtain the supernatant. Mix the supernatant with the Ni-NTA filler that has been equilibrated with buffer A in advance, and bind for 1 hour. Collect the filler and wash the filler with buffer A in large quantities until no impurities are washed out. Then add buffer B to the filler to elute Dda. The eluted Dda is passed through a desalting column equilibrated with buffer C for buffer exchange.
  • step 3.5 Remove the centrifuge tube from the magnetic rack and centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube.
  • step 4.5 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 4.4 into the metal bath preheated at 25°C in step 4.1 for ligation reaction and set the timer to 30 minutes.
  • step 5.5 Remove the centrifuge tube from the magnetic rack and centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube.
  • T7 Exonuclease T7 exonuclease; NEB, M0263LVIAL
  • NEB NE Buffer TM 4
  • step 4.5 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 4.4 into the metal bath preheated at 25°C in step 4.1 for ligation reaction and set the timer to 30 minutes.
  • step 5.5 Remove the centrifuge tube from the magnetic rack and centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de construction d'une banque de séquençage, une banque de séquençage, un procédé de séquençage et un kit de construction d'une banque de séquençage. Le procédé de construction d'une banque de séquençage comprend : la réalisation, sous l'action d'une exonucléase, d'un traitement de digestion sur un échantillon à tester qui est lié à un lieur, de façon à obtenir une banque de séquençage cible.
PCT/CN2022/142020 2022-12-26 2022-12-26 Procédé de réduction de lieurs libres dans une banque de séquençage Ceased WO2024138320A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2022/142020 WO2024138320A1 (fr) 2022-12-26 2022-12-26 Procédé de réduction de lieurs libres dans une banque de séquençage
CN202280102106.5A CN120344725A (zh) 2022-12-26 2022-12-26 减少测序文库中游离接头的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/142020 WO2024138320A1 (fr) 2022-12-26 2022-12-26 Procédé de réduction de lieurs libres dans une banque de séquençage

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WO2024138320A1 true WO2024138320A1 (fr) 2024-07-04

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090275087A1 (en) * 2008-04-30 2009-11-05 Gi Mikawa Asymmetric adapter library construction
CN105899678A (zh) * 2013-10-18 2016-08-24 牛津纳米孔技术公司 经修饰的酶
US20170073748A1 (en) * 2015-09-16 2017-03-16 PetaOmics, Inc. Methods and compositions for genomic target enrichment and selective dna sequencing
CN107794575A (zh) * 2017-10-16 2018-03-13 深圳华大基因股份有限公司 用于Pacbio平台的DNA大片段文库构建方法和试剂盒
CN109957605A (zh) * 2019-03-20 2019-07-02 嘉兴菲沙基因信息有限公司 适用于PacBio测序平台的海洋动物与软体动物的建库测序方法
CN111534504A (zh) * 2014-01-22 2020-08-14 牛津纳米孔技术公司 将一个或多个多核苷酸结合蛋白连接到靶多核苷酸的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090275087A1 (en) * 2008-04-30 2009-11-05 Gi Mikawa Asymmetric adapter library construction
CN105899678A (zh) * 2013-10-18 2016-08-24 牛津纳米孔技术公司 经修饰的酶
CN111534504A (zh) * 2014-01-22 2020-08-14 牛津纳米孔技术公司 将一个或多个多核苷酸结合蛋白连接到靶多核苷酸的方法
US20170073748A1 (en) * 2015-09-16 2017-03-16 PetaOmics, Inc. Methods and compositions for genomic target enrichment and selective dna sequencing
CN107794575A (zh) * 2017-10-16 2018-03-13 深圳华大基因股份有限公司 用于Pacbio平台的DNA大片段文库构建方法和试剂盒
CN109957605A (zh) * 2019-03-20 2019-07-02 嘉兴菲沙基因信息有限公司 适用于PacBio测序平台的海洋动物与软体动物的建库测序方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENG JIA, GUO PEI-XUAN: "Membrane-embedded Channel of Bacteriophage phi29 DNA-packaging Motor for Single Molecule Sensing and Nanomedicine", CHINESE BULLETIN OF LIFE SCIENCES, vol. 23, no. 11, 30 November 2011 (2011-11-30), pages 1114 - 1129, XP009555905, ISSN: 1004-0374 *

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