WO2022131285A1 - Ｄｎａサンプルのシーケンスにおけるアダプター結合効率を評価する方法 - Google Patents

Ｄｎａサンプルのシーケンスにおけるアダプター結合効率を評価する方法 Download PDF

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WO2022131285A1
WO2022131285A1 PCT/JP2021/046211 JP2021046211W WO2022131285A1 WO 2022131285 A1 WO2022131285 A1 WO 2022131285A1 JP 2021046211 W JP2021046211 W JP 2021046211W WO 2022131285 A1 WO2022131285 A1 WO 2022131285A1
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dna
adapter
reaction
sequence
molecule
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French (fr)
Japanese (ja)
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正史 田中
英俊 猪子
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GENODIVE PHARMA Inc
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GENODIVE PHARMA Inc
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Priority to CA3201748A priority Critical patent/CA3201748A1/en
Priority to AU2021401369A priority patent/AU2021401369A1/en
Priority to KR1020237021963A priority patent/KR20230121076A/ko
Priority to CN202180083883.5A priority patent/CN116615537A/zh
Priority to US18/267,732 priority patent/US20240102089A1/en
Priority to EP21906647.9A priority patent/EP4265721A4/en
Priority to JP2022570032A priority patent/JPWO2022131285A1/ja
Publication of WO2022131285A1 publication Critical patent/WO2022131285A1/ja
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/125—Electrophoretic separation

Definitions

  • the present invention relates to a method for evaluating adapter binding efficiency in order to optimize adapter binding conditions in a DNA sequence using a double-stranded barcode adapter.
  • it is useful for DNA sequencing of samples containing only rare DNA samples such as liquid biopsy.
  • cfDNA cell-free DNA released from tissue cells due to apoptosis or the like
  • blood also contains cfDNA derived from cancer cells.
  • cfDNA derived from cancer cells In recent years, research on the use of cfDNA derived from cancer cells for cancer diagnosis has progressed, and it has been attracting attention as a minimally invasive test method called "liquid biopsy".
  • cfDNA derived from cancer cells contained in the peripheral blood of a cancer patient is sequenced using a so-called next-generation sequencer, and the presence or absence of a mutation peculiar to the cancer cell is detected easily. Cancer can be diagnosed with minimal invasiveness. However, it is said that 1 mL of blood contains only about 1000 molecules of cfDNA corresponding to the human genome, regardless of whether it is a healthy person or a cancer patient. In the case of cancer patients, only a part of them is cfDNA derived from cancer cells. In order to sequence using such a rare DNA sample, it is necessary to improve the accuracy of the sequence as much as possible.
  • next-generation sequencer is widely used as a device used for sequencing DNA samples.
  • error occurrence rate in the next-generation sequencer currently used is about 0.1%.
  • Molecular barcode technology can be mentioned as a method for improving reading accuracy in next-generation sequencers such as massively parallel sequencers.
  • a molecular bar code generation sequence is added to a target gene region of DNA to perform PCR amplification. Since the base sequences having the same molecular barcode are derived from the same molecule, the reading error can be eliminated by creating the consensus sequence.
  • Patent Document 1 As a method of adding a barcode sequence to cfDNA, there are a method using a primer and a method of adding an adapter in which a barcode generation sequence is embedded by ligation.
  • a Y-type adapter containing a specific molecular index (UMI) that is, a barcode-generating sequence is added by ligation to both ends of a double-stranded DNA fragment, and PCR amplification is performed to cause errors, noise, etc. It is described that it enables a sequence with excellent accuracy and sensitivity without being affected by the above.
  • Patent Document 2 proposes a new Y-type (fork-type) adapter with a reduced error rate.
  • DNA ligase an enzyme that catalyzes the reaction reaction between a DNA fragment and an adapter.
  • reaction conditions recommended by the raw material (enzyme) supplier are adopted, and the reaction efficiency is also high. There is no simple way to measure.
  • the present invention is to easily and accurately evaluate the binding efficiency in order to optimize the conditions for binding (ligating) a Y-shaped adapter to both ends of a double-stranded DNA fragment in a sequence of DNA samples.
  • the challenge is to provide the method of.
  • the present inventors prepared a model DNA fragment (double strand) and a model Y-type adapter, and intentionally prepared a model-binding molecule in which the adapter was bound to 1 to 4 positions at 4 ends of the model DNA fragment. Furthermore, it was verified for the first time that the prepared model-binding molecule can be classified (identified) according to the number of adapter bonds by electrophoresis. Based on these findings, the present inventors determined the reaction efficiency of the binding (ligation) between the DNA fragment to be analyzed in the DNA sequence and the Y-type adapter, and the degree of mobility of the generated binding molecule in electrophoresis. Established a method of evaluation by. The evaluation method can be used to optimize the conditions of the ligation reaction using DNA ligase.
  • the present invention is a method for evaluating ligation reaction efficiency in which Y-type adapters are ligated to both ends of the analysis target DNA in a sequence of analysis target DNA using a Y-type adapter.
  • the reaction efficiency is evaluated by electrophoresis of the reaction mixture containing the binding molecule between the DNA generated by the ligation reaction under predetermined conditions and the Y-type adapter, and analyzing the separated bands based on the number of adapters bound to the DNA. Provide a way to do it.
  • the present invention evaluates the first reaction efficiency by (1) carrying out a ligation reaction under a first predetermined condition and carrying out the above-mentioned evaluation method for the reaction mixture, and then (2) the above-mentioned first.
  • the ligation reaction was carried out under the second predetermined condition in which at least a part of the predetermined conditions of 1 was changed, and the second reaction efficiency was evaluated by carrying out the above-mentioned evaluation method for the reaction mixture, and (3).
  • a method for optimizing the conditions of a ligation reaction which comprises comparing the first reaction efficiency with the second reaction efficiency. In this optimization method, the optimum reaction conditions can be found by repeating the above steps (2) and (3) a plurality of times.
  • the reaction efficiency in the reaction of binding (ligating) a Y-type adapter to a DNA molecule to be analyzed can be evaluated by a simple operation. Therefore, by carrying out the evaluation method of the present invention while changing the reaction conditions, it is possible to easily and accurately find the conditions for completely binding the Y-type adapter to the DNA molecule with high efficiency.
  • the evaluation method of the present invention is effective in sequencing using liquid biopsy, but in sequencing genomes and DNA, it is essential to sequence after creating a library. Therefore, the library creation technique using the evaluation method of the present invention can be applied not only to liquid biopsy but also to general sequencing of genomes and DNA, and contributes to the efficiency, accuracy and accuracy of sequencing. It is a thing.
  • next-generation sequencer when the DNA to be analyzed is sequenced using a Y-type adapter, as shown in FIG. 1, Y-type is formed at both ends of the DNA to be analyzed (double strand). Connect (ligate) the adapter. At this time, with respect to the upper DNA strand and the lower DNA strand of the DNA to be analyzed, if the adapter molecules corresponding to both ends of each DNA strand are appropriately bound (ligated) via a phosphodiester bond, PCR amplification is performed. Then, the sequence information of the DNA strand can be obtained. However, the PCR reaction does not proceed on the DNA strand to which the adapter molecule is not properly bound, and appropriate sequence information cannot be obtained.
  • NGS next-generation sequencer
  • the number shown in parentheses in FIG. 2 is the number of adapter molecules bound to the DNA molecule. That is, a DNA molecule (0) to which an adapter is not bound at all, a DNA molecule (1) in which one adapter molecule is bound to only one end of one DNA strand, and an adapter molecule to which only one end of both DNA strands is bound.
  • the present inventors have a DNA molecule in which four adapters are completely bound ((4) in FIG. 2; “complete adapter-binding molecule”) and an adapter binding is incomplete.
  • Model molecules corresponding to various DNA molecules ((0), (1), (2-1), (2-2), (2-3) and (3) in FIG. 2; "incomplete adapter-binding molecule”). was produced.
  • a model DNA double chain and a model Y-type adapter as shown in FIG. 3 were created.
  • a model DNA duplex having different 5'overhang sequences at both ends ( ⁇ and ⁇ in FIG. 3A) and a 5'overhang sequence matching each overhang sequence of the model DNA duplex.
  • Two types of Y-type adapters with ( ⁇ 'and ⁇ 'in FIG. 3B) were created.
  • each overhang sequence an asymmetrical sequence, the bonds between molecules are limited to predetermined bonds.
  • the binding products produced in a solution containing a model DNA double chain and two types of model Y-type adapters are limited to binding molecules in which one of the model Y-type adapters is added to both ends of the model DNA double chain. No binding occurs between model DNA double chains or model Y-type adapters. Furthermore, by setting the specific 5'overhang end to P or OH, it is possible to predefine the presence or absence of coupling at the 5'overhang end.
  • the model DNA double strand was prepared by PCR.
  • the PCR chain length can be set arbitrarily, but it was set to 170 bp, which is the average chain length of cfDNA.
  • a recognition sequence for a TypeIIS restriction enzyme such as BsaI or BbsI was added to the primer used in PCR. Therefore, it is possible to generate a preset 5'overhang end by cleaving the PCR product with these enzymes.
  • a PCR product having a BsaI site at one end and a BbsI site at the other end is prepared, and if necessary, a 5'overhang end generated by BsaI and a 5'overhang generated by BbsI. Either or both ends were converted to OH ends by post-kinase dephosphorylation.
  • the created model DNA double strand is only the molecule (A1) having a phosphate group at the 5'end of both the upper and lower strands of the model DNA molecule (A0) and the 5'end ( ⁇ part) of the upper strand.
  • model Y type adapter had an adapter sequence for an Illumina sequencer, and a phosphorylated 5'overhang end was synthesized.
  • the model Y-type adapter is a Y-type adapter consisting of two DNA strands that can partially hybridize with each other, and the 5'end of the hybridizable portion is over.
  • the overhang portion has a base sequence ( ⁇ ') complementary to the overhang portion ( ⁇ ) of the model DNA molecule, or a base sequence ( ⁇ ) complementary to the overhang portion ( ⁇ ) of the model DNA molecule.
  • ⁇ ' base sequence complementary to the overhang portion ( ⁇ ) of the model DNA molecule.
  • model Y-type adapters (B1-1 and B1-2) having a phosphate group at the 5'end of the overhang portions ( ⁇ 'and ⁇ ') were synthesized.
  • model Y-type adapters can be created by synthesizing and annealing single-stranded DNA.
  • the presence or absence of a phosphodiester bond depending on the presence or absence of a phosphoric acid group can be specified by preparing an adapter having a hydroxyl group at the 5'end, in addition to the above-mentioned dephosphorylation method after enzymatic cleavage.
  • the samples containing the above-mentioned various bound molecules were electrophoresed using an electrophoresis device, and the mobility (mobility) of each bound molecule was compared.
  • the mobility of these molecules by electrophoresis is complicatedly affected by the migration conditions including the molecular weight of each molecule, the shape of the molecule, the properties of the carrier for electrophoresis, etc., and in general, the mobility of each molecule is determined in advance. It is considered difficult to foresee.
  • FIG. 5 shows.
  • the degree of mobility varies depending on the number and position of the coupled adapters. That is, it can be separated based on the difference in the type of binding molecule (complete adapter binding molecule and incomplete adapter binding molecule) generated by the ligation reaction, in other words, the binding reaction of the Y-type adapter to the DNA molecule to be analyzed. It was verified that the efficiency of the above can be measured with high accuracy by electrophoresis.
  • Example 1 A DNA double chain / adapter binding reaction that imitated the actual NGS library creation process was performed, and its efficiency was measured.
  • the target cfDNA is mainly a double chain of about 170 bp, but its terminal is generated as a result of a reaction by a DNase in vivo, so that it has a 5'-overhang, a 3'-overhang, or a blunt end. It is believed that there is.
  • the end of the Y-type adapter molecule for creating a library for NGS generally has a structure in which T protrudes at the 3'end.
  • a kit for creating a library for NGS generally consists of two modules, a module for repairing the end of a DNA duplex and adding dA to the 3'end, and a module for adapter ligation. Since the efficiency of binding between the DNA duplex and the adapter molecule in each kit and each reaction condition is the result of three types of reactions by both modules, the model DNA duplex in the model binding experiment of this example is one end. A 170 bp DNA having a 4-base 5'-overhang and a 4-base 3'-overhang at the other end was used.
  • This model DNA duplex consists of a PCR reaction with a primer having a 4-base 5'-overhang restriction enzyme recognition sequence and a primer having a 4-base 3'-overhang restriction enzyme recognition sequence, followed by PCR product restriction. Created by enzymatic treatment.
  • the Y-type adapter itself included in the commercially available kits (manufactured by company A and company B) actually used for creating the NGS library was used.
  • Their structure is "Y-type partial double chain", and basically only one type of Y-type adapter is included in each kit, and the end of the double chain part is a 3'overhang of 1 base dT. Has a structure.
  • the ratio of the fully adapter-bound molecule to the generated bound molecule under each condition shown in FIG. 6 was as follows.
  • Example 2 Using cfDNA contained in liquid biopsy, a ligation reaction of a Y-type adapter was carried out under the following conditions.
  • -CfDNA used A DNA sample eluted in purified water using Qiagen's QIAamp Circulating Nucleic Acid from plasma separated within 48 hours after blood was collected in the Streck blood collection tube.
  • -Reaction conditions The reaction kit uses NEBNext Ultra II DNA Library Prep Kit for Illumina manufactured by NEB): 1.
  • 1. cfDNA Repair 50 ng in 30 microliters, add dT 2.
  • the above reaction product is mixed with a 75 picomoll Y-type adapter (IDT), and a ligation reaction is carried out in a total of 52.5 microliters.
  • the reaction temperature is 7 degrees and the reaction time is 12 hours.
  • FIG. 7 shows the results of electrophoresis of the bound molecule generated by the above ligation reaction. Under the above conditions, it was confirmed that complete coupling of the adapter could be achieved with high efficiency (70% or more).

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PCT/JP2021/046211 2020-12-15 2021-12-15 Ｄｎａサンプルのシーケンスにおけるアダプター結合効率を評価する方法 Ceased WO2022131285A1 (ja)

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CA3201748A CA3201748A1 (en) 2020-12-15 2021-12-15 Method for evaluating adapter ligation efficiency in sequencing of dna sample
AU2021401369A AU2021401369A1 (en) 2020-12-15 2021-12-15 Method for evaluating adaptor ligation efficiency in sequencing DNA sample
KR1020237021963A KR20230121076A (ko) 2020-12-15 2021-12-15 Dna 샘플의 시퀀스에 있어서의 어댑터 결합 효율을평가하는 방법
CN202180083883.5A CN116615537A (zh) 2020-12-15 2021-12-15 评价dna样品的测序中的适配体结合效率的方法
US18/267,732 US20240102089A1 (en) 2020-12-15 2021-12-15 Method for Evaluating Adapter Ligation Efficiency in Sequencing of DNA Sample
EP21906647.9A EP4265721A4 (en) 2020-12-15 2021-12-15 METHOD FOR EVALUATING ADAPTER LIGATION EFFICIENCY IN A DNA SAMPLE SEQUENCE
JP2022570032A JPWO2022131285A1 (https=) 2020-12-15 2021-12-15

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WO2025117738A1 (en) * 2023-11-28 2025-06-05 Illumina, Inc. Methods of improving unique molecular index ligation efficiency

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JP2019504624A (ja) 2016-01-29 2019-02-21 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 核酸配列決定のための新規アダプターおよび使用法
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JP2019504624A (ja) 2016-01-29 2019-02-21 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 核酸配列決定のための新規アダプターおよび使用法
JP2020516281A (ja) * 2017-04-14 2020-06-11 ガーダント ヘルス， インコーポレイテッド 試料核酸にアダプターを付着する方法

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Publication number Priority date Publication date Assignee Title
WO2025117738A1 (en) * 2023-11-28 2025-06-05 Illumina, Inc. Methods of improving unique molecular index ligation efficiency

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