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WO2024138517A1 - Conception d'adaptateurs de banque pour améliorer le débit de séquençage - Google Patents

Conception d'adaptateurs de banque pour améliorer le débit de séquençage Download PDF

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Publication number
WO2024138517A1
WO2024138517A1 PCT/CN2022/143317 CN2022143317W WO2024138517A1 WO 2024138517 A1 WO2024138517 A1 WO 2024138517A1 CN 2022143317 W CN2022143317 W CN 2022143317W WO 2024138517 A1 WO2024138517 A1 WO 2024138517A1
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Prior art keywords
exonuclease
sequencing
sample
tested
connector
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Ceased
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PCT/CN2022/143317
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English (en)
Chinese (zh)
Inventor
师虓
王欧
郭斐
章文蔚
董宇亮
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to CN202280102166.7A priority Critical patent/CN120344663A/zh
Priority to PCT/CN2022/143317 priority patent/WO2024138517A1/fr
Publication of WO2024138517A1 publication Critical patent/WO2024138517A1/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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • 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 present invention belongs to the field of biotechnology, and specifically, relates to a method for establishing a sequencing library, and more specifically, relates to a sequencing library, a sequencing method, and a kit for constructing a sequencing library or sequencing.
  • Nucleic acid sequencing has become an indispensable and important research method in the field of life science research.
  • the accompanying genomic technology based on large-scale sequencing data has also played an important role in different research and application directions, such as tracing the causes of complex diseases and dynamically monitoring their development process, targeted breeding of economic crops and animals, and research and protection of different biological genetic resources.
  • 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.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the present invention proposes a method for establishing a sequencing library.
  • the method comprises: digesting the sample to be tested connected with a connector under the action of a nuclease to obtain the sequencing library.
  • the 5' end of the connector does not have a phosphorylation modification, and the 3' end of the connector is connected to the 5' end of the sample to be tested.
  • the method of the embodiment of the present invention on the one hand, by introducing a nuclease to degrade the fragment to be tested that is not completely connected, the product of the incomplete connection of the connector, and the unconnected connector, the number of abnormal sequencing is reduced, and the sequencing accuracy and sequencing throughput are improved.
  • the connector by using a connector without phosphorylation modification at the 5' end and treating the connected product with a nuclease, the connector itself is prevented from connecting to form a dimer, and the free connector that is not connected to the two ends of the inserted fragment to be tested is digested into a single strand, thereby reducing the residual connector sequence that may be sequenced or reducing the time it occupies the sequencing channel, and improving the sequencing throughput.
  • the chain without phosphorylation modification at the 5' end will be further degraded by the nuclease, exposing the binding position of the anchor sequence (since the 5' end cannot be connected to the 3' end of another single chain when it is not phosphorylated, it will be recognized and digested by the nuclease), and the anchor sequence itself will not be covalently bound to the library to be tested, after the motor protein passes through the anchor sequence binding position, the anchor sequence will be stripped from the sequencing library itself, thereby realizing the reuse of the anchor sequence and improving the library capture efficiency.
  • the above method may further include at least one of the following additional technical features:
  • the sample to be tested connected with the connector has a gap, and the gap is located between the 5' end of the connector and the 3' end of the sample to be tested.
  • 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 helicase moves along the DNA in a direction from the 5' end to the 3' end, and the exonuclease is a 5'->3' exonuclease.
  • the 5’->3’ exonuclease recognizes double-stranded DNA
  • the exonuclease includes at least one selected from T7 exonuclease, T5 exonuclease, Lambda exonuclease, Exonuclease VI, and Exonuclease VIII (truncated).
  • the exonuclease 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 further includes performing anti-degradation modification on the 5' end and/or 3' end of the sample to be tested connected with a connector.
  • some exonucleases that can recognize the 5' end of single-stranded DNA and digest 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 with a 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 connector is a Y-type connector or a non-Y-type connector.
  • 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 sample to be tested connected with the connector is obtained by:
  • a first purification treatment is further included for the A addition treatment product.
  • the process further includes performing a second purification process on the ligation product.
  • the sample to be tested is a DNA sample.
  • 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 further provides a sequencing method.
  • the method comprises mixing the above-mentioned sequencing library with an anchor sequence, wherein at least a portion of the anchor sequence is complementary to at least a portion of a strand of the adapter.
  • the mixed product is subjected to sequencing to obtain a sequence of the sample to be tested.
  • the sequencing is performed on a nanopore sequencing platform.
  • This method can be used to efficiently determine the sequence information of nucleic acid samples, and has high sensitivity, high accuracy, good repeatability, and high sequencing throughput.
  • the present invention also provides a kit for constructing a sequencing library or sequencing.
  • the kit includes a reagent, and the reagent includes a nuclease exonuclease for digesting the sample to be tested connected with a connector.
  • the inventors have found that by using the kit of the present invention, combined with the above-mentioned method for establishing a sequencing library, sequencing library, and sequencing method, a high-quality sequencing library can be obtained, and it can be effectively applied to a high-throughput sequencing platform, thereby determining the nucleic acid sequence information of the nucleic acid sample, and the information obtained has high accuracy and a large sequencing throughput.
  • the kit further comprises at least one of the following additional technical features:
  • the reagent further includes a first reagent, and the first reagent is suitable for fragmenting the sample to be tested.
  • the reagent further includes a second reagent, and the second reagent is suitable for performing end repair and A addition treatment on the sample to be tested.
  • the reagent further includes a third reagent, and the third reagent is suitable for performing a connector connection process on the sample to be tested.
  • the kit further comprises a linker, and the 5' end of the linker is not phosphorylated.
  • the nuclease includes at least one of T7 exonuclease, Lambda exonuclease, T5 Exonuclease, Exonuclease VI, Exonuclease VIII (truncated), Exonuclease III, Exonuclease IX, and Exonuclease X.
  • the kit further comprises an anchor sequence, at least a portion of which is complementary to at least a portion of one strand of the adapter.
  • the anchor sequence can be connected to one strand of the adapter through complementary base pairing, thereby bringing the sequence to be tested connected with the adapter to the vicinity of the nanopore sequencing hole for sequencing.
  • FIG1 is a diagram showing the Pacbio cyclization consensus sequencing principle according to an embodiment of the present invention.
  • FIG2 is a flow chart of library construction containing an exonuclease step according to an embodiment of the present invention
  • FIG3 is a diagram of a sequencing process using a 5' unphosphorylated adapter and a 5'->3' exonuclease according to an embodiment of the present invention
  • FIG5 is a comparison diagram of library bands before and after enzyme digestion according to an embodiment of the present invention.
  • FIG6 is a comparison result of the number of single-channel sequencing signals after library construction and sequencing with and without phosphorylation of the connector 5' according to an embodiment of the present invention.
  • the present invention provides a method for establishing a sequencing library.
  • the method comprises the following steps: initial nucleic acid molecule fragmentation (optional), end repair & A addition, adapter ligation, nuclease treatment, anchor sequence annealing.
  • initial nucleic acid molecule fragmentation optionally
  • end repair & A addition end repair & A addition
  • adapter ligation nuclease treatment
  • anchor sequence annealing anchor sequence annealing.
  • the DNA sequence of the genome is selectively interrupted to obtain fragmented genomic DNA molecules, so as to improve the library capture rate when sequencing on the machine.
  • the fragmented genomic DNA molecules are end-repaired and "A" is added.
  • the genomic DNA after adding "A” is subjected to product purification treatment, the purpose is to obtain a purer sample, so as to increase the accuracy and sequencing throughput during sequencing.
  • the sample obtained after purification is connected to the adapter.
  • the adapter connection refers to mixing the adapter with T4DNA ligase (NEB, E6057AVIAL) to finally obtain the sample after the adapter connection.
  • the adapter connection sample is subjected to product purification, and the sample with higher purity is extremely important for the accuracy and data volume of the sequencing on the machine.
  • the purified connection product is first digested under the action of a nuclease to degrade the fragment to be tested that has not been connected at all, a chain in the free adapter that is not connected to the two ends of the inserted fragment to be tested, and the product of incomplete adapter connection, thereby reducing the time occupied by the undesired library for the sequencing channel time, thereby improving the sequencing throughput.
  • the exonuclease can be selected from at least one of T7 exonuclease, T5 exonuclease, Lambda exonuclease, Exonuclease VI, and Exonuclease VIII (truncated).
  • the sample treated with the exonuclease is subjected to product purification to obtain a preliminary library sample.
  • the obtained preliminary library sample is co-incubated with the helicase Dda protein to finally obtain a library that can be used for sequencing.
  • the direction of movement of the helicase along the DNA is from the 5' end to the 3' end.
  • the method can be used to efficiently prepare sequencing samples, and the obtained sequencing library can be effectively applied to a high-throughput sequencing platform, thereby effectively determining the nucleic acid sequence information of the library sample.
  • the method for preparing a sequencing library of the present invention is simple in process, extremely easy to operate, and the operation process is extremely easy to standardize and promote, and is low in cost, high in sensitivity, high in accuracy, and good in repeatability.
  • the library of the sample to be tested is obtained by the above method for establishing a sequencing library.
  • a sequencing library is constructed using a 5' unphosphorylated adapter and a 5'->3' exonuclease, and the library sample to be tested is placed on a nanopore sequencing platform for on-machine sequencing.
  • the anchor sequence captures the sequencing library near the nanopore for sequencing.
  • Motor protein refers to a type of protein distributed inside or on the surface of cells, which is responsible for the macroscopic movement of a part of the substance in the cell or the entire cell.
  • primer used in this article is a general term for oligonucleotides that can complementarily pair with a template and synthesize a DNA chain complementary to the template under the action of DNA polymerase.
  • Primers can be natural RNA, DNA, any form of natural nucleotides, or even non-natural nucleotides such as LNA or ZNA.
  • iSpC3 is a three-hydrocarbon carbon chain that is often used as a spacer in oligonucleotide chains.
  • iSp18 is an 18-atom-long hexaethylene glycol chain that is commonly used as a spacer in oligonucleotide chains.
  • Exonucleases are a class of enzymes that degrade nucleotides one by one from the end of a polynucleotide chain. According to the specificity of the enzyme to the secondary structure of the substrate, it can be divided into three categories: 1 Exonucleases that act on single strands, such as E. coli exonuclease I and E. coli exonuclease VII. 2 Exonucleases that act on double strands, such as E. coli exonuclease III, bacteriophage exonucleases, and T7 bacteriophage gene VI exonuclease.
  • 1 Exonucleases that act on single strands such as E. coli exonuclease I and E. coli exonuclease VII.
  • 2 Exonucleases that act on double strands such as E. coli exonuclease III, bacteriophage exonucleases, and
  • the linker sequence is obtained by annealing chemically synthesized SEQ ID NO.1 and SEQ ID NO.2.
  • the specific implementation method is as follows:
  • the helicase Dda (amino acid sequence is shown in SEQ ID NO.5) is prepared by recombinant expression in Escherichia coli, and the helicase is used as a motor protein.
  • Buffer A 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;
  • Buffer B 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;
  • Buffer C 20 mM Tris-HCl pH 7.5, 50 mM NaCl;
  • Buffer D 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;
  • 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.
  • thrombin Yishen Bio, 20402ES05
  • the mixed sample to the ssDNA cellulose (Sigma, D8273-10G) filler equilibrated with buffer C, and digest and bind overnight at 4°C. Collect the ssDNA cellulose filler, wash it with buffer C 3-4 times, and then elute it with buffer D.
  • the protein concentrate purified from the ssDNA cellulose is passed through the molecular sieve Superdex 200 (Sigma, GE28-9909-44), and the molecular sieve buffer used is buffer E.
  • the target protein was collected, concentrated, and frozen.
  • the concentration of the purified protein was quantified using Nanodrop.
  • the purity of the protein was tested by HPLC and SDS-PAGE electrophoresis at the same time. The results are shown in Figure 4.
  • This example takes the construction and purification of the Dda and Ad3 complex as an example, and the specific steps are as follows:
  • the buffer solution used in this step is prepared as follows:
  • Buffer H 50 mM Tris (pH 7.5), 2.5 M NaCl;
  • Buffer I 50 mM Tris (pH 7.5), 2.5 M NaCl, 24% PEG8000, 0.05% TWEEN20;
  • Buffer J 50 mM Tris (pH 7.5), 2.5 M NaCl, 20% PEG 8000;
  • Buffer K 50 mM Tris (pH 7.5), 20 mM NaCl.
  • Magnetic beads (VAHTSTM DNA Clean Beads #N411-03) were used to purify and remove free proteins, crosslinkers, and non-specific binding non-target complexes in the reaction system.
  • the specific operation steps are as follows: first take the magnetic beads out of the refrigerator in advance, shake and mix, and then place them at room temperature. First, wash the magnetic beads with Buffer H (magnetic bead washing buffer) so that the buffer in the magnetic beads is replaced with the appropriate pH high salt buffer used in the purification conditions.
  • Buffer H magnetic bead washing buffer
  • Buffer I magnetic bead equilibration buffer
  • Buffer J magnetic bead equilibration buffer
  • 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 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 6.4 Place the 1.5 mL DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) in step 6.3 in the metal bath preheated at 37°C in step 6.1 for ligation reaction and set the timer to 5 minutes.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

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Abstract

La présente invention concerne un procédé pour établir une banque de séquençage, une banque de séquençage, un procédé de séquençage et un kit pour la construction d'une banque de séquençage ou pour le séquençage. Le procédé d'établissement d'une banque de séquençage comprend : la digestion, sous l'action d'une exonucléase, d'un échantillon à l'essai connecté à un adaptateur pour obtenir une banque de séquençage. L'extrémité 5' de l'adaptateur ne présente pas de modification de phosphorylation, et l'extrémité 3' de l'adaptateur est reliée à l'extrémité 5' dudit échantillon.
PCT/CN2022/143317 2022-12-29 2022-12-29 Conception d'adaptateurs de banque pour améliorer le débit de séquençage Ceased WO2024138517A1 (fr)

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CN202280102166.7A CN120344663A (zh) 2022-12-29 2022-12-29 提升测序通量的文库接头设计
PCT/CN2022/143317 WO2024138517A1 (fr) 2022-12-29 2022-12-29 Conception d'adaptateurs de banque pour améliorer le débit de séquençage

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WO2016058134A1 (fr) * 2014-10-14 2016-04-21 深圳华大基因科技有限公司 Élément de liaison et son procédé d'utilisation pour construire une banque de séquençage
WO2016082129A1 (fr) * 2014-11-26 2016-06-02 深圳华大基因研究院 Procédé et réactif pour la construction d'une banque cyclique d'acide nucléique simple brin à double séquence de liaison
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
CN112105744A (zh) * 2018-05-24 2020-12-18 牛津纳米孔科技公司 方法
CN113736778A (zh) * 2021-09-14 2021-12-03 成都齐碳科技有限公司 测序接头、构建方法、纳米孔建库试剂盒及应用
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WO2016058134A1 (fr) * 2014-10-14 2016-04-21 深圳华大基因科技有限公司 Élément de liaison et son procédé d'utilisation pour construire une banque de séquençage
WO2016082129A1 (fr) * 2014-11-26 2016-06-02 深圳华大基因研究院 Procédé et réactif pour la construction d'une banque cyclique d'acide nucléique simple brin à double séquence de liaison
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
CN112105744A (zh) * 2018-05-24 2020-12-18 牛津纳米孔科技公司 方法
CN113736778A (zh) * 2021-09-14 2021-12-03 成都齐碳科技有限公司 测序接头、构建方法、纳米孔建库试剂盒及应用
CN114854826A (zh) * 2022-05-13 2022-08-05 北京齐碳科技有限公司 序列、包含序列的接头及其用途

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KE SUN, CHANGJIAN ZHAO, XIAOJUN ZENG, YUEJIA CHEN, XIN JIANG, XIANTING DING, LU GOU, HAIYANG XIE, XINQIONG LI, XIALIN ZHANG, SHENG: "Active DNA unwinding and transport by a membrane-adapted helicase nanopore", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, vol. 10, no. 1, 1 December 2019 (2019-12-01), XP055747456, DOI: 10.1038/s41467-019-13047-y *

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