WO2023137667A1 - Lieur et son utilisation dans la construction d'une banque de dnb - Google Patents

Lieur et son utilisation dans la construction d'une banque de dnb Download PDF

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Publication number
WO2023137667A1
WO2023137667A1 PCT/CN2022/073026 CN2022073026W WO2023137667A1 WO 2023137667 A1 WO2023137667 A1 WO 2023137667A1 CN 2022073026 W CN2022073026 W CN 2022073026W WO 2023137667 A1 WO2023137667 A1 WO 2023137667A1
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Prior art keywords
linker
adapter
nucleic acid
library
primer
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PCT/CN2022/073026
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English (en)
Chinese (zh)
Inventor
王业钦
杨林
欧日晶
杨贵芳
吕硕
聂自豪
张韶红
张艳艳
陈芳
Original Assignee
深圳华大智造科技股份有限公司
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Priority to PCT/CN2022/073026 priority Critical patent/WO2023137667A1/fr
Publication of WO2023137667A1 publication Critical patent/WO2023137667A1/fr

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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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

Definitions

  • the invention belongs to the field of bioengineering, and in particular relates to a linker and its application in constructing a DNB library.
  • PCR amplification process which is highly enriched and highly adaptable to micro-sample and hotspot SNPs, and can obtain high-quality data at a very low cost.
  • PCR Free PCR-free amplification
  • the second is PCR-free amplification (PCR Free), which avoids errors introduced by amplification and can obtain real sample information.
  • the complete PCR Free technology has strict sample requirements, cumbersome steps and high cost, which compresses people's desire for it.
  • the current library construction technology based on the DNBseq platform is classified according to whether it is amplified or not.
  • the technical problem to be solved by the present invention is to provide a linker and its application, especially the application in rapid construction of a single-stranded circular library.
  • the present invention develops a novel linker and a corresponding method for library construction and sequencing.
  • the library construction and sequencing method is simpler, can more truly reflect the original information of samples, and can meet the needs of this field with higher cost performance.
  • the present invention mainly solves the above-mentioned technical problems through the following technical solutions.
  • the present invention provides a linker, which comprises linker 1, linker 2 and anchor primers; wherein the nucleic acid sequences at both ends of the anchor primers are complementary to part of the nucleic acid sequences of linker 1 and linker 2, respectively.
  • the adapter 1 and the adapter 2 preferably both contain one or more tag sequences, reverse complementary sequences and sequencing primer binding sequences; and the 5' end of the adapter has phosphorylation modification, and the 3' end locks and exposes at least one T base after annealing with the reverse complementary sequence at the 5' end;
  • the two ends of the anchor primer respectively contain capture sequences complementary to adapter 1 and adapter 2, and a single-stranded nucleic acid structural sequence.
  • the adapter 1 also contains 1 or 2 index primer binding sequences.
  • the linker 2 also contains 1 or 2 index primer binding sequences.
  • both the adapter 1 and the adapter 2 further contain 1 or 2 index primer binding sequences.
  • the binding sequence of the index primer and the binding sequence of the sequencing primer are limiting factors of the sequencing platform, and can be adjusted accordingly according to the sequencing platform or bioinformatics analysis.
  • the reverse complementary sequences inside the adapter 1 and the adapter 2 are 5-10 bp locked.
  • the linker 1 and the linker 2 can respectively anneal to form a hairpin structure through the reverse complementary sequence under appropriate conditions.
  • both the length of the linker and the length range of the anchor primer can be adjusted to meet the needs of the actual library construction scenario.
  • the length of the anchor primer is determined according to the length of the inserted library.
  • the anchor primer is a single-stranded nucleic acid sequence longer than 35 bp, for example, the length can be 35-200 bp, such as 75 bp; the capture sequence length of the anchor primer is 15-25 bp, and the single-stranded structural sequence length of the anchor primer is designed according to the length of the library fragment to be tested.
  • the single-stranded structural sequence of the anchor primer is 35-55 bp.
  • the present invention also provides a kit for constructing a sequencing library, which comprises adapter 1, adapter 2 and anchor primers as defined above.
  • the kit further comprises a polymerase.
  • the molar ratio of the adapter 1, the adapter 2 and the anchor primer is 1:1:1.
  • the kit also includes other solutions routinely used in this field, such as 5 ⁇ STE Buffer and NF Water, etc.
  • the kit is usually prepared in the form of serial linker complex dilutions during use.
  • the preparation method of the adapter complex dilution comprises the following steps:
  • the conditions of the reaction described in step (1) are preferably: 95°C for 5min, 70°C for 5min, 45°C for 5min, and 25°C for 5min.
  • the condition of reaction described in step (2) is preferably: 25 °C 15min.
  • the present invention also provides a method for constructing a sequencing library, the method comprising:
  • Adapter complex preparation mix the adapter 1 and adapter 2 defined above with the anchor primer and incubate to obtain the adapter complex;
  • step (1) (2) performing a ligation reaction between the nucleic acid fragment to be detected and the linker complex in step (1), the ligation reaction further comprising a ligase;
  • the nucleic acid fragment is selected from: nucleic acid fragments obtained by genome disruption, PCR products, free nucleic acid fragments and reverse transcription products.
  • the present invention also provides a method for simultaneously detecting the sense strand and the antisense strand of nucleic acid, which comprises:
  • a new adapter complex structure ( Figure 1), which is composed of tag adapter 1, tag adapter 2 and anchor primers, can capture DNA fragments with phosphorylated 5-terminals and cohesive ends with A bases at the 3-terminal for library construction;
  • Index adapter 1 (adapter 1, Figure 2): This adapter is composed of one or more tags, index primer binding sequence, a complementary structure and sequencing primer binding sequence. Its 5' end has phosphorylation modification, and its 3' end is locked after refolding and a T base is exposed for pairing connection;
  • Adapter 2 (adapter 2, Figure 3): This adapter is composed of one or more tags, tag primer binding sequence, a complementary structure and sequencing primer binding sequence. Its 5' end has phosphorylation modification, and its 3' end is locked after refolding and a T base is exposed for pairing connection;
  • An anchor primer contains two nucleic acid sequences complementary to adapter 1 and adapter 2 respectively, and a single-stranded nucleic acid sequence, which can simultaneously capture and anchor two adapters correspondingly designed to form an arched structure, and can be used as a primer for the subsequent preparation of DNA nanoballs;
  • PCR Free format can be selected for library building, which has good compatibility and independent selectivity; PCR links can be exempted for better quality samples, and PCR amplification steps can be reduced for poor quality and trace samples; low sample size requirements, good compatibility, and high accuracy;
  • the double-strand combined library construction and sequencing method can completely retain the original double-strand information of the sample, maintain the authenticity of the sample information to the greatest extent, and expand new applications for special needs.
  • Figure 1 is a schematic diagram of the connection of the novel linker complex.
  • Figure 2 is the connector adapter 1.
  • Figure 3 is the connector adapter2.
  • Figure 4 is the connector Link.
  • Figure 5 shows the database building process
  • Figure 6 is a graph showing the size distribution of fragmented DNA products.
  • Fig. 7 is an electrophoresis diagram of the product after verification and amplification of DNB.
  • Figure 8 is a 6 ⁇ GAPDH Primer mix gel electrophoresis image.
  • Step 1 Pre-synthesize three primers of adapter 1, adapter 2 and Link and dilute them into 100 ⁇ L dilution using TE (Sangon, Cat. No. B548106-0500). Anneal the Adapter 1 primer dilution and Adapter 2 primer dilution. The annealing method is "take Adapter 1 (100 ⁇ M)/12.5 ⁇ L+Adapter 2 (100 ⁇ M)/12.5 ⁇ L, 5 ⁇ STE Buffer (BGI)/3 ⁇ L, and place it on a PCR instrument for reaction.
  • the program is “25 °C for 15 min, 4 °C storage’” to obtain the linker dilution (25 ⁇ M) required for this method.
  • linker dilution solution 25 ⁇ M/8 ⁇ L, 10 ⁇ Phi29Buffer (Thermo, product number B62)/40 ⁇ L, NF Water (Invitrogen, product number AM9932)/152 ⁇ L and mix to prepare Make DNB buffer for later use.
  • Step 2 Fragment the DNA sample using Hieff NGS OnePot DNA Fragmentation Reagent ( ⁇ , Cat. No. 13474ES96).
  • the processing method is "take sample DNA/150ng, OnePot mix/7.5 ⁇ L, NF water UP to 30 ⁇ L; 30°C for 10min, 65°C for 20min, and store at 4°C (heat lid set at 70°C)".
  • the processed sample DNA was taken out and centrifuged for a simple purification.
  • the operation was "take the reaction product of the previous step and add 27 ⁇ L (0.9 ⁇ ) magnetic beads (VAZYME, product number N411-03), mix well and place at room temperature for 5 minutes; then place it on a magnetic stand for adsorption for 2 minutes, and remove the supernatant; add 80% ethanol to rinse once and dry until the surface of the magnetic beads is rough; add 22 ⁇ L TE (Sanko, product number B548106-0500) Mix and elute, and keep the supernatant; take 1 ⁇ L and use Qubit dsDNA HS Assay Kit (Invitrogen, product number Q32854) to quantify the concentration, and the total yield is required to be greater than 30ng for future use.
  • Step 3 DNB preparation before sequencing, the operation is "take 20 ⁇ L of the Make DNB buffer prepared in step 1 and mix with 30 ng of the product obtained in step 2, then add DNB polymerase I (MGI, 1000004803)/40 ⁇ L, T4 DNA ligase (repaid) (enzymatic, product number L6030-HC-L) 1 ⁇ L, make up to 80 ⁇ L with NF Water, 2 5°C for 10 minutes, stored at 4°C (heated lid set at 35°C); after the reaction, the reaction solution was taken out and centrifuged, and DNB polymerase II (MGI, 1000017261)/4 ⁇ L was added, 30°C for 25 minutes, stored at 4°C (total volume 84 ⁇ L, heated lid set at 35°C)".
  • DNB polymerase I MMI, 1000004803
  • T4 DNA ligase repaid
  • Step 1 Prepare Make DNB buffer according to the method of step 1 in Experimental Method 1.
  • Step 2 Perform PCR amplicon library preparation.
  • the first combination is 5-terminal phosphorylated F primer and R primer, template and Taq enzyme (TaKaRa, Cat. No. R001A), and the second combination is conventional F primer and R primer, template, Taq enzyme, and T4 polynucleotide kinase (Thermo, Cat. No. EK0031).
  • the preparation method is "dilute each F primer and R primer to 25 ⁇ M, and mix them in equimolar amounts at a ratio of 1:1 for later use; take the mixed primer/4 ⁇ L, 2 ⁇ M ⁇ Ltiplex PCR Readymix (BGI, catalog number 1000014386)/25 ⁇ L, sample/1ng, NF Water/UP to 50 ⁇ L; the amplification program is 95°C/3min, 1 cycle; 95°C/ 20s, 60°C/20s, 72°C/30s, 30 cycles; 72°C/10min, one cycle; 12°C storage".
  • This process can be optimized according to its own situation, and no specific requirements are required, but it is necessary to ensure that the product is fully extended so that A is added to the end to facilitate ligation.
  • the processed sample DNA was taken out and centrifuged and purified once.
  • the operation was as follows: take the reaction product from the previous step and add 40 ⁇ L (0.8 ⁇ ) magnetic beads, mix well and place at room temperature for 5 minutes; then place it on a magnetic stand for 2 minutes and remove the supernatant; add 80% ethanol to rinse once and discard the supernatant; add 80% ethanol to rinse once and dry until the surface of the magnetic beads is rough; Assay Kit quantitative concentration, the total output must be greater than 30ng for use.
  • Step 3 DNB preparation before sequencing, according to the situation described in step 2, we need to prepare in two ways.
  • the amplicon library prepared by the first combination we can directly prepare it according to step 4 in method 1.
  • the second combination we need to change the steps to "take 20 ⁇ L of the Make DNB buffer prepared in step 1 and mix it with 30 ng of the product obtained in step 3, then add DNB polymerase I/40 ⁇ L, T4 DNA ligase (repaid) 1 ⁇ L, T4 polynucleotide kinase 1 ⁇ L, make up to 80 ⁇ L with NF Water, 30°C for 15 minutes, and store at 4°C (heat lid set at 35 degrees); Centrifuge, add DNB polymerase II/4 ⁇ L, store at 30°C for 25 minutes, and store at 4°C (total volume 84 ⁇ L, heat lid set at 35°C)”. After the reaction, take 2 ⁇ L and use the Qubit ssDNA Assay Kit for quantification. If the concentration is greater than
  • F1 CAGAACGACATGGCTACGATCCGACGT (SEQ ID NO: 16)
  • R1 TGCATGGCGACCTTATCAGAGTTA (SEQ ID NO: 17)
  • GAPDH-1F CCCCGGTTTCTATAAATTGAGC (SEQ ID NO: 4)
  • GAPDH-1R CGCTTGGCCTCCGACTTGAACTCACCCGTTGACTCCG (SEQ ID NO:5)
  • GAPDH-2F CTCCTCTGCGACACGTGA (SEQ ID NO: 6)
  • GAPDH-2R CGCTTGGCCTCCGACTTGTTCTCTCCCCTCCGCGCA (SEQ ID NO: 7)
  • GAPDH-3F CAGGAGGTCCCTACTCCCG (SEQ ID NO: 8)
  • GAPDH-3R CGCTTGGCCTCCGACTTGAGAATAATCTAGGAAAAGCATCACCCG (SEQ ID NO:9)
  • GAPDH-4F CCCAATCCTCCCGGTGACAT (SEQ ID NO: 10)
  • GAPDH-4R CGCTTGGCCTCCGACTTATGGGTGGAGTCGCGTGT (SEQ ID NO: 11)
  • GAPDH-5F ATCAATGACCCCTTCATTGA (SEQ ID NO: 12)
  • GAPDH-5R CGCTTGGCCTCCGACTTGATCTCGCTCCTGGAAGAT (SEQ ID NO: 13)
  • GAPDH-6F TAAGCAGTTGGTGGTGCA (SEQ ID NO: 14)
  • GAPDH-6R CGCTTGGCCTCCGACTTCTTCACCACCATGGAGAAG (SEQ ID NO: 15)
  • GAPDH-1F 5phos-CCCCGGTTTCTATAAATTGAGC (SEQ ID NO: 4)
  • GAPDH-1R 5phos-CGCTTGGCCTCCGACTTGAACTCACCCGTTGACTCCG (SEQ ID NO:5)
  • GAPDH-2F 5phos-CTCCTCTGCGACACGTGA (SEQ ID NO: 6)
  • GAPDH-2R 5phos-CGCTTGGCCTCCGACTTGTTCTCTCCCCTCCGCGCA (SEQ ID NO: 7)
  • GAPDH-3F 5phos-CAGGAGGTCCCTACTCCCG (SEQ ID NO:8)
  • GAPDH-3R 5phos-CGCTTGGCCTCCGACTTGAGAATAATCTAGGAAAAGCATCACCCG (SEQ ID NO:9)
  • GAPDH-4F 5phos-CCCAATCCTCCCGGTGACAT (SEQ ID NO: 10)
  • GAPDH-4R 5phos-CGCTTGGCCTCCGACTTATGGGTGGAGTCGCGTGT (SEQ ID NO: 11)
  • GAPDH-5F 5phos-ATCAATGACCCCTTCATTGA (SEQ ID NO: 12)
  • GAPDH-5R 5phos-CGCTTGGCCTCCGACTTGATCTCGCTCCTGGAAGAT (SEQ ID NO: 13)
  • GAPDH-6F 5phos-TAAGCAGTTGGTGGTGCA (SEQ ID NO: 14)
  • GAPDH-6R 5phos-CGCTTGGCCTCCGACTTCTTTCACCACCATGGAGAAG (SEQ ID NO: 15)
  • the primers used in this protocol were all synthesized by Jinweizhi (Suzhou).
  • step 2 Carry out the experiment according to the experimental method one.
  • step 2 three 150ng DNA samples were processed respectively, and the concentration and total amount of the obtained products were shown in Table 1, which met the requirements.
  • Agilent 2100 high sensitivity
  • the interrupted product obtained in the previous step was prepared as DNB according to step 3.
  • the concentration is shown in Table 2, which meets the requirements of the machine.
  • a pair of quality inspection primers F1/R1 was used to amplify the DNB, and electrophoresis was performed on a 1% agarose gel to check that the size of the amplified product was in line with expectations ( FIG. 7 ).
  • the library was sequenced using the MGI-2000 platform and supplemented with the data of a routine library construction (MGIEasy Universal DNA Library Preparation Kit) for comparison, and the data GC_Content and Mapping_Rate were analyzed using BWA; Raw_Q30, Raw_Reads, Clean_Reads and Clean_Rate were analyzed using Soapnuke, and the results are shown in Table 3.
  • the test was carried out according to the second experimental method. Six pairs of amplification primers were mixed and then amplified. Three parallel replicates were performed for samples with different concentrations. The PCR products were electrophoresed on 1% agarose gel and compared with Maker 100 (TIANGEN, Cat. No. MD109).
  • MGI-2000 platform uses the MGI-2000 platform to sequence the library and supplement the data of a conventional process library (MGIEasy Universal DNA Library Preparation Kit) for comparison, use BWA to analyze the data GC_Content and Mapping_Rate; use Soapnuke to analyze Raw_Q30, Raw_Reads, Clean_Reads and Clean_Rate, and the results are shown in Table 6.

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Abstract

Lieur et son utilisation pour la construction d'une banque DNB. Le lieur comprend un lieur 1, un lieur 2 et une amorce d'ancrage ; les séquences d'acide nucléique à deux extrémités de l'amorce d'ancrage sont respectivement complémentaires des séquences d'acide nucléique partielles du lieur 1 et de l'amorce d'ancrage 2. La construction d'une banque de DNB à l'aide du lieur permet de préparer une banque en une seule étape et présente un meilleur rapport coût/efficacité que d'autres procédés ; en outre, le lieur peut s'adapter à des échantillons de différentes charges et les formes d'adaptation sont diversifiées ; le lieur peut également être utilisé pour capturer de l'ADN double brin et effectuer un séquençage double brin.
PCT/CN2022/073026 2022-01-20 2022-01-20 Lieur et son utilisation dans la construction d'une banque de dnb WO2023137667A1 (fr)

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