WO2019090482A1 - Procédé de construction de bibliothèque de séquençage à haut rendement de seconde génération - Google Patents

Procédé de construction de bibliothèque de séquençage à haut rendement de seconde génération Download PDF

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WO2019090482A1
WO2019090482A1 PCT/CN2017/109770 CN2017109770W WO2019090482A1 WO 2019090482 A1 WO2019090482 A1 WO 2019090482A1 CN 2017109770 W CN2017109770 W CN 2017109770W WO 2019090482 A1 WO2019090482 A1 WO 2019090482A1
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polynucleotide
tail
substrate
tailing
dna
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PCT/CN2017/109770
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Chinese (zh)
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陈文浩
郭焕焕
王小庆
梁丽丽
陈琼
权胜卯
井忠英
常璐媛
张翼
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北京优乐复生科技有限责任公司
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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
    • 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 relates to methods and kits for the construction of second generation high throughput sequencing libraries, and more particularly to methods and kits for the construction of high throughput sequencing libraries based on terminal transferases.
  • the second-generation sequencing technology has a faster sequencing speed and higher throughput, which is in line with the current scientific and technological development requirements for sequencing.
  • the second-generation sequencing technology platforms include Illumina's Hiseq, Miseq, Nextseq, Novaseq, and Life Technologies' SOLID system, PGM, Proton, and others.
  • the technical idea of the second-generation sequencing technology is to synthesize and sequence, that is, to determine the DNA sequence according to the signal changes brought by the newly synthesized different bases.
  • the Illumina sequencing platform detects the light signal
  • the Life sequencing platform detects the acid-base change. The current changes.
  • second-generation sequencing technologies become more mature, their clinical applications will become more widespread.
  • Circulating DNA also known as free DNA
  • free DNA is DNA that exists outside the cell in the blood.
  • the main source of free DNA is apoptotic cells or bone marrow cells, and the DNA released by these cells is cleaved by nuclease in vivo to produce a small fragment DNA of about 166 bp in length (Y.M. Dennis Lo et al. Science Translational Medicine. 2010. 10: 61ra91).
  • Free DNA is in a state of dynamic equilibrium in the body, so free DNA can be an important parameter for health assessment. Changes in tumorigenesis, organ transplantation, etc. can lead to changes in the properties of free DNA in peripheral blood. These properties include the length of free DNA, base information, and apparent modification. Therefore, free DNA can be used as an early diagnosis, monitoring, and prognostic evaluation of disease. An important marker of non-invasive detection.
  • Methylation sequencing libraries need to be constructed prior to methylation sequencing of free DNA using second-generation high-throughput sequencing technology.
  • the second generation of high-throughput methylation sequencing libraries is constructed by pre-library construction, including terminal fill-in, 5'-end phosphorylation, 3' end-suspension A and linker ligation steps; After the bisulfite treatment, the bisulfite treatment causes a large amount of DNA damage, and the template that can be finally sequenced accounts for less than 10% of the original template (Masahiko Shiraishi et al. 2004. 10: 409-415).
  • the construction process of the methylation sequencing library needs 1) each step needs to be purified, and the operation is cumbersome; 2) the filling step will artificially introduce nucleotides and change the true methylation state; 3) a large amount of DNA template in hydrogen sulfite It is destroyed during salt treatment and is lost after PCR amplification. Therefore, it is necessary to develop a better method of building a library, which can reduce the damage of DNA damage caused by bisulfite.
  • the present invention provides a method of tailing a deoxypolynucleotide substrate; and further provides a A second generation high throughput sequencing library construction method.
  • the method of the present invention is applicable not only to normal DNA, but also to samples with severe damage such as FFPE samples, ancient DNA, and DNA samples after bisulfite treatment.
  • the present invention provides a method of tailing a deoxypolynucleotide substrate, the method comprising the steps of: (1) mixing the deoxypolynucleotide substrate with a substance to form a first mixture: a) dGTP or dCTP nucleotide; b) terminal deoxynucleotidyl transferase; c) tail control component comprising a polynucleotide homopolymer of 5 to 20 nucleotides in length (abbreviated as 5b-20b) a tail-control region, wherein the polynucleotide homopolymer is complementary to a) nucleotide; (2) incubating the first mixture, a tailing reaction occurs at the 3' end of the deoxynucleotide substrate, at the substrate 3' The dGTP or dCTP polynucleotide is added to form a 3' tailing region of the substrate.
  • the deoxypolynucleotide substrate is a double-stranded or single-stranded deoxynucleotide sequence; preferably a single-stranded deoxypolynucleotide substrate; preferably the polynucleotide homopolymer of the tail-control region is poly(dC) Homopolymer.
  • the polynucleotide of the tail-control region is a heteropolymer sequence composed of dC and rC bases.
  • the present invention provides a method for directly performing complementary strand synthesis on a tailed deoxypolynucleotide substrate, the method comprising: adding a ribonuclease RNase HII degradation after the tailing reaction of the step (2)
  • the ribonucleotide in the polynucleotide homopolymer or the tail-controlling molecule of the tail-controlling component, the 3'-end free hydroxyl group is generated, and the tail-tailing component is used as a template for the complementary strand with the substrate having the 3' tailing region as a template
  • Extending adding DNA polymerase and deoxynucleotide (including dATP, dTTP, dCTP and dGTP) to the first mixture to form a second mixture using step (3); and incubating the second step in step (4) a mixture, a nucleotide polymerization reaction occurs at the 3' end of the tail-control component complementary to the substrate after the tailing, synthesis A complementary
  • a further embodiment of the present invention provides a method for synthesizing a complementary strand (hereinafter also referred to as "dual-chain synthesis") in a manner of strand displacement extension for a tailed deoxypolynucleotide substrate: step (3) -1): after the tailing reaction of the step (2), adding an extension primer, a DNA polymerase and a deoxynucleotide complementary to the substrate 3' tailing region to the first mixture to form a second mixture
  • Step (4) incubating the second mixture, performing nucleotide polymerization at the 3' end of the extension primer to synthesize a complementary strand of the substrate to obtain a double-stranded deoxypolynucleotide; and step (5): from the The double-stranded deoxypolynucleotide is isolated in the second mixture.
  • the two-chain synthesis is carried out by a degradative extension method, which first degrades the tail-control molecules in the tail-control component, and then adds the complementary 3' tailing region to the substrate.
  • the primers, DNA polymerase and deoxynucleotides are extended to form a second mixture.
  • a ligation step of adding a 5' sequencing linker to a double-stranded nucleotide substrate and step (6): adding 5' to the isolated double-stranded deoxypolynucleotide
  • the linker and ligase are sequenced to form a third mixture, the third mixture is incubated, and the double-stranded deoxypolynucleotide is ligated to the 5' sequencing linker.
  • kits comprising: a deoxynucleotide substrate, a dGTP or dCTP nucleotide, a terminal deoxynucleotidyl transferase, and a tail-control component, wherein the tail-control group
  • the polynucleotide comprises a polynucleotide homopolymer of 5 to 20 nucleotides in length that is complementary to a dGTP or dCTP nucleotide.
  • the kit can be used to control the tailing reaction of substrate nucleotides.
  • kit of the present invention further comprises a DNA ligase or an RNA ligase.
  • the kit of the present invention further comprises a DNA polymerase, and a deoxynucleotide comprising dATP, dTTP, dCTP and dGTP.
  • kit of the present invention further comprises an extension primer.
  • kit of the present invention further comprises at least one of RNase, USER enzyme and nicking enzyme.
  • kit of the invention further comprises a 5' sequencing linker.
  • the present invention effectively controls the length of the terminal transferase tailing at the 3' end of the polynucleotide substrate by designing a tail-control component. Further, the inventors have also found that the polynucleotide tail of the polynucleotide substrate can be linked to the linker while forming a double-stranded polynucleotide structure by annealing hybridization at a certain temperature with a poly region previously added to the tail-control component. By this method, the sequence of interest can be added very efficiently at the 3' end of the polynucleotide substrate, for example, the sequence of interest can be an priming sequence for sequencing sequencing for next generation sequencing.
  • the present invention also finds that a terminal transferase adds a dGTP or dCTP core to a polynucleotide substrate.
  • glycosidic acid forms a poly(dG) or (dC) tail
  • the substrate utilization efficiency is significantly higher than the addition of dATP or dTTP nucleotides to form a poly(dA) or (dT) tail.
  • the nucleic acid is denatured into a single strand, and after the substrate is tailed, the linker is ligated, and then the complementary strand extension of the polynucleotide substrate is completed, optionally the 3' end dA tail of the complementary strand is ligated, and the 5' linker is ligated. Enrichment, resulting in a library for next-generation sequencing.
  • the library construction process can construct a whole genome methylation sequencing library for genomic DNA of as low as 10 ng human culture cell source, and obtain efficient sequencing results.
  • FIG. 4 Schematic diagram of 5' sequencing linker
  • Figure 5 Results of the addition of nucleotide homo-tails to a mixed single-stranded DNA polynucleotide substrate with different bases at the 3' end using TdT enzyme
  • Figure 6 Results of the addition of nucleotide homo-tails to a mixed single-stranded DNA polynucleotide substrate containing a partial random sequence and having different bases at the 3' end using the TdT enzyme
  • Figure 8 Fragment distribution results of methylated sequencing libraries constructed by controlled tailing of bisulfite-treated lambda DNA by TdT enzyme by polynucleotide homopolymer
  • Figure 9 Experimental results of using a tail-controlling molecule to control the length of a poly(dG) tail added to a substrate by a TdT enzyme and attaching the poly(dG) tail of the substrate to the linker under the action of a ligase
  • Figure 10 Experimental results of studying the effect of reaction time on the attachment of a substrate to a tail after the addition of a poly(dG) tail
  • Figure 11 Fragment distribution results of a methylation sequencing library constructed by two-chain synthesis by degradation and strand displacement; wherein, Figure 11A and Figure 11B are respectively methylation sequencing libraries prepared by degradation and strand displacement. Fragment distribution.
  • Figure 12 Fragment distribution results of a methylation sequencing library constructed by controlled tailing of bisulfite-treated ⁇ -DNA by TdT enzyme using a tail-tailing component with a stem-loop structure
  • Figure 13 Fragment distribution results of a methylated sequencing library constructed by controlled tailing of bisulfite-treated ⁇ -DNA by TdT enzyme using tail-control components with different lengths of tail-controlling regions
  • Figure 13A shows the fragment distribution results of the methylation sequencing library prepared by the tail-control component with a length of 5b poly(dC).
  • Figure 13B shows the fragment distribution results of a methylation sequencing library prepared by a tail-control component with a tail length of 6b poly(dC).
  • Figure 13C shows the fragment distribution results of a methylation sequencing library prepared by a tail-control component with a length of 7b poly(dC).
  • Figure 13D shows the fragment distribution results of a methylation sequencing library prepared by a tail-control component with a tail length of 8b poly(dC).
  • Figure 13E shows the fragment distribution results of a methylation sequencing library prepared by a tail-control component with a tail length of 9b poly(dC).
  • Figure 13F shows the fragment distribution results of a methylation sequencing library prepared by a tail-control component with a length of 10b poly(dC).
  • Figure 13G shows the fragment distribution results of a methylation sequencing library prepared by controlling the tail component of the 11b poly(dC)
  • Figure 13H shows the fragment distribution results of a methylation sequencing library prepared by controlling the tail component of the length 12b poly(dC).
  • Figure 13I shows the fragment distribution results of a methylation sequencing library prepared by the tail-control component with a length of 13b poly(dC).
  • Figure 13J shows the fragment distribution results of a methylation sequencing library prepared by a tail-control component with a length of 20b poly(dC).
  • Figure 14 Fragment distribution results of a human genome methylation sequencing library constructed based on the method of the present invention and a conventional method; wherein, Fig. 14A and Fig. 14B are respectively a human genome methylation sequencing library constructed based on the method of the present invention and a conventional method; Fragment distribution results.
  • Polynucleotide substrates are polynucleotide substrate fragments that require tailing reactions and/or library construction.
  • the polynucleotide substrate is single stranded or double stranded DNA.
  • the polynucleotide substrate is a chemically treated nucleotide sequence including, but not limited to, a bisulfite treated polynucleotide.
  • the polynucleotide substrate can be of natural origin or synthetic.
  • a natural source is a polynucleotide sequence from a prokaryote or eukaryote, such as a human, mouse, virus, plant or bacterium.
  • the polynucleotide substrate of the present invention may also be a severely damaged sample such as FFPE sample, ancient DNA, or bisulfite-treated DNA.
  • the polynucleotide substrate is tailed and can be used in assays involving microarrays and to generate libraries for next generation nucleic acid sequencing.
  • the tailed polynucleotide substrate can also be used for efficient cloning of polynucleotide sequences.
  • the polynucleotide substrate is single stranded or double stranded and comprises a 3' terminal free hydroxyl group. In some In aspect, the polynucleotide substrate is double stranded and comprises blunt ends. In other aspects, the double stranded polynucleotide substrate comprises a 3' recessed end.
  • the length of the protruding or recessed end of the polynucleotide substrate can vary. In various aspects, the length of the protruding or recessed end of the polynucleotide substrate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.
  • the polynucleotide substrate is between about 10 and about 5000 nucleotides in length, or between about 40 and about 2000 nucleotides, or between about 50 and about Between 1000 nucleotides, or between about 100 and about 500 nucleotides. In a further aspect, the polynucleotide substrate is at least 3 up to about 50, 100 or 1000 nucleotides in length.
  • the present invention controls the tail length and efficiency of the polynucleotide substrate by the addition of a tailing component.
  • the tail-control component comprises a tail-control region consisting of a polynucleotide homopolymer of dGTP or dCTP, for example, the tail-control region may be a polynucleotide homopolymer of dGTP or dCTP of 5-13 nucleotides in length.
  • Polynucleotide homopolymers are polynucleotide chains joined by the same nucleotide.
  • the tailing region of the invention is preferably a poly(dC) or poly(dG) nucleotide homopolymer sequence; and a heteropolymer sequence comprising: (i) dC and rC nucleotides, or (ii) dG With rG nucleotides.
  • the polynucleotide homopolymer of the tail-control region of the invention has a length of 5-20 nucleotides, preferably 7-20, 9-20 nucleotides, further preferably 5-10 nucleotides, 7-10 nucleotides, more preferably 7-9 nucleotides.
  • a certain length of dGTP or dCTP polynucleotide homopolymer can effectively control the tailing of the polynucleotide substrate to about 20 nucleotides.
  • the tailing component is a polynucleotide homopolymer, that is, the tailing component comprises only the tailing zone.
  • the tail-controlling component is a tail-controlling molecular sequence joined by a tail-control region and an X-region (also referred to as an X-region sequence, or a sequencing linker sequence of a tail-control component), such as the invention.
  • X-region sequence also referred to as an X-region sequence, or a sequencing linker sequence of a tail-control component
  • SEQ ID NO: 11 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18.
  • SEQ ID NO: 19 and SEQ ID NO: 20 are examples of SEQ ID NO: 20.
  • an "X region sequence” provides a priming sequence for amplification or sequencing of a nucleic acid fragment, or a marker sequence for distinguishing between different substrate molecules, and in some aspects for next generation sequencing applications.
  • the X region sequence may be, but is not limited to, a Next Generation Sequencing (NGS) linker sequence compatible with Illumina, Ion Torrent, Roche 454 or SOLiD sequencing platforms, such as the Illumina Truseq shown in Table 7. Library sequence.
  • the X region sequence can be a DNA sequence, an RNA sequence or a hybrid sequence comprising DNA and RNA.
  • the tail-control component is a tail-control zone and an X-zone, and a linker sequence that is complementary to the X-region (see Figure 1), and the tail-control component is also referred to as a "tail-control connector".
  • a linker having only a sequence complementary to the X region is Called “short link", as shown in Table 2.
  • a linker sequence including an extended primer binding region is referred to as a "long linker" as shown in Table 3.
  • the tail-control component may be the same polynucleotide molecule, and the polynucleotide forms a stem-loop structure to generate a partially double-stranded polynucleotide in which the X region is complementary to the linker sequence. In this stem-loop configuration, the polynucleotide is the same
  • the polymer moiety is a single molecule; the tail-control component can also be two separate polynucleotide molecules that hybridize to each other, ie, the linker sequence is a polynucleotide molecule capable of complementing the X region sequence.
  • the tail-control component of the present invention is preferably a double-stranded tail-control component selected from Table 2 or Table 3.
  • the polynucleotide of the tailing component comprises a peptide nucleic acid, a Schizophyllan polysaccharide, a locked nucleic acid, and combinations thereof.
  • the invention provides compositions wherein the tailing component is single stranded or at least partially double stranded.
  • “Partially double-stranded” means that the tail-control component comprises a single-stranded portion and a double-stranded portion.
  • the partially double stranded tailing component is hybridized to the linker molecule by the X region sequence of the tail control molecule (in some embodiments, the linker sequence is The NGS linker sequence is produced, and a partial sequence of the tail-controlling molecule is complementary to the linker molecule.
  • hybridization occurs within a single tail-control molecule that forms a hairpin structure, such that the tail-control component that forms part of the double-stranded structure can be either a single molecule or a multi-molecular structure.
  • the tailing component comprises a blocking group.
  • a blocking group as used herein is a moiety that prevents extension by an enzyme. If there is no blocking group, the enzyme is able to synthesize the polynucleotide by adding nucleotides. Blocking groups include, but are not limited to, phosphate groups, carbon triarms, dideoxynucleotides, ribonucleotides, amino groups, and reverse deoxythymidine.
  • the 5' end of the tailing component linker is phosphorylated, with a blocking group at the 3' end and a 3' blocking group at the tailing region.
  • the term "tailing” as used herein may be interchanged with the term “controlled tailing.”
  • the present invention provides a method of tailing a deoxypolynucleotide substrate for adding a desired amount of dGTP or dCTP nucleotide to the 3' end of the polynucleotide substrate in a controlled manner.
  • the tail-control component comprises a polynucleotide homopolymer of 5 to 20 nucleotides in length, a newly added nucleoside of the tail-control component and the substrate.
  • the acid and polytail sequences form a double-stranded structure, thus reducing the rate of polymerization and allowing the tail of the polynucleotide substrate to be controlled over a range of lengths (see Figure 2).
  • the TdT enzyme is used to control the TdT enzyme to add a poly(dG) tail at the 3' end of the polynucleotide substrate (also known as a nucleus) using a tail-control component comprising a poly(dC) nucleotide homomeric sequence. Glycosylate (dG) homopolymeric tail). Further, the substrate of the polynucleotide The poly(dG) tail is linked to the linker of the tail-control component to form the 3' end plus tail of the substrate.
  • the tail-control component comprises 5-20, preferably 5-13, further preferably 7-10, more preferably 7-9 poly(dC)-containing nucleotide homopolymers Sequence of objects.
  • the molar concentration ratio of the polynucleotide substrate to the tail-control component ranges from 1:1 to 1:100, preferably from 1:5 to 1:50.
  • the pH of the tailing reaction ranges from about 5.0 to about 9.0; the molar ratio of polynucleotide substrate to mononucleotide ranges from 1:10-1:20000, preferably 1:100-1: 2000; incubation time is from 1 minute to 120 minutes, preferably from 0.5 to 60 minutes, from 0.5 to 30 minutes, from 1 to 20 minutes, from 1 to 15 minutes or from 1 to 10 minutes.
  • the extension reaction is further carried out using the deoxypolynucleotide substrate as a template (see the library construction of FIG. 2 and the two-chain synthesis of FIG. 3).
  • RNase is added to degrade the polynucleotide homopolymer of the tailing component to produce a 3' hydroxyl group; DNA polymerase and deoxynucleotide are added, and the deoxynucleotide base is Incubate with the tail control component.
  • This method generates a nucleotide extension reaction at the 3' end of the tail-control component to synthesize a complementary strand of the deoxypolynucleotide substrate to obtain a double-stranded deoxypolynucleotide.
  • the incubation time for the extension reaction is from 1 minute to 60 minutes, preferably from 1 to 30 minutes, from 1 to 20 minutes, from 1 to 15 minutes, or from 1 to 10 minutes.
  • the present invention first degrades the tail-controlling molecule, and then adds the extension primer to synthesize the double-stranded deoxypolynucleotide using the polynucleotide substrate as a template.
  • the tail-control molecule comprises a ribonucleotide and is incubated with a ribonuclease (RNase) under conditions of sufficient activity thereof, followed by incubation at a temperature above 80 ° C. The substrate is separated.
  • RNase ribonuclease
  • the ribonuclease is selected from at least one of RNase H, RNase HII, RNase A, and RNase T1.
  • the tail control molecule comprises dU nucleotides and can be incubated by dU glycosylation enzyme followed by incubation at a temperature above 80 ° C, or with dU glycosylase and depurination/depyrimidine nucleic acid A mixture of endonucleases, such as incubation with the USER enzyme, is degraded.
  • the double-stranded region of the tail-control component comprises a specific sequence recognizable by a nicking endonuclease, and can be cleaved by a nicking enzyme such as Nt.BspQI, followed by a nick at 80 ° C or higher. Incubation at temperature separates the tail molecules from the substrate.
  • an extension primer is added, and the tail molecule is separated from the substrate polynucleotide substrate by strand displacement, and the extension reaction is carried out using the deoxypolynucleotide substrate as a template.
  • the method comprises: after the tailing reaction, adding an extension primer complementary to the 3' tailing region of the substrate, a DNA polymerase and a deoxynucleotide, and incubating with the substrate nucleotide substrate; 3' of the extension primer A nucleotide extension reaction occurs at the end to synthesize a complementary strand of the substrate to obtain a double-stranded deoxypolynucleotide.
  • the DNA polymerase has strand displacement activity, for DNA aggregation Under the condition that the strand displacement activity of the synthase and the DNA polymerization activity are sufficient, the tail-control molecule can be separated from the polynucleotide substrate by strand displacement while completing the extension reaction.
  • DNA polymerases with strand displacement activity useful in the practice of this patent include, but are not limited to, large fragment Bst DNA polymerase, Bst 3.0 DNA polymerase, Klenow large fragment, phi29 DNA polymerase.
  • the tailing molecule polynucleotide in the tailing component is first degraded, and the extension primer is added for extension reaction.
  • the method comprises: after the tailing reaction, adding RNase to degrade the tail-controlling polynucleotide in the tail-control component, and then adding an extension primer complementary to the 3' tailing region of the substrate, DNA polymerase and deoxynucleotide Incubation with a substrate nucleotide substrate; a nucleotide extension reaction occurs at the 3' end of the extension primer to synthesize a complementary strand of the substrate to obtain a double-stranded deoxy polynucleotide.
  • the DNA polymerase in the extension reaction has 3'-5' exonuclease activity (corrected activity), resulting in a blunt-ended double-stranded structure upon extension; in other aspects, the DNA polymerase lacks 3 '-5' exonuclease activity (corrected activity), after the extension is completed, a double-stranded structure in which dA is prominent is obtained.
  • DNA polymerases useful in the present patent include, but are not limited to, the following species or combinations thereof: full length Bst DNA polymerase, KAPA high fidelity hot start DNA polymerase, KAPA high fidelity hot start Uracil+ DNA polymerase, Bst 3.0DNA polymerase, Phusion TM High fidelity DNA polymerase, Hot Start Taq DNA polymerase, Ex Taq DNA polymerase, Deep Vent R TM DNA polymerase, T4 DNA polymerase, Klenow large fragment.
  • the 5' sequencing linker provides priming sequences for amplification or sequencing of nucleic acid fragments and is used in some aspects for next generation sequencing applications (see Figure 4).
  • the 5' sequencing linker is formed by annealing two polynucleotide strands.
  • the 5' linker sequence is selected from, but not limited to, a Next Generation Sequencing (NGS) linker sequence compatible with Illumina, Ion Torrent, Roche 454 or SOLiD sequencing platforms, such as the Illumina Truseq library sequence shown in Table 7.
  • the linker sequence may be a DNA sequence, an RNA sequence or a heteropolymer sequence comprising DNA and RNA, such as the 5' sequencing linker shown in Table 4.
  • the 5' sequencing linker is ligated with the double-stranded polynucleotide substrate obtained after the extension reaction of step (4), and the structure of the terminus can be either a blunt end or a sticky end.
  • Sticky ends include, but are not limited to, dt sticky ends.
  • the double-stranded deoxypolynucleotide undergoes a ligation reaction with the 5'-sequencing linker.
  • the 5' linker is only joined to the 3' end of the complementary strand of the polynucleotide substrate.
  • the 5' linker is only joined to the 5' end of the polynucleotide substrate.
  • the 5' linker is joined to the 5' end of the polynucleotide substrate and the 3' end of the complementary strand joining the polynucleotide substrate.
  • a dA cohesive end is added to the 3' end of the polynucleotide substrate.
  • the DNA polymerase lacking 3'-5' exonuclease activity (correcting activity) is used to continue adding the dA cohesive ends after completion of the extended duplex reaction.
  • the DNA polymerase having 3'-5' exonuclease activity separates the substrate from the DNA polymerase after the blunt end is generated after completion of the extended strand reaction, and then the lack of 3 The '-5' exonuclease activity (corrected activity) of the DNA polymerase and the nucleotide are tailed, and a sticky end is added.
  • a DNA polymerase lacking 3'-5' exonuclease activity (correcting activity) can be, but is not limited to, Klenow (deficient 3'-5' exonuclease activity), Taq DNA polymerase.
  • Ligase enzymes useful in the methods of the invention may be DNA ligases and RNA ligases including, but not limited to, T4 DNA ligase, E. coli DNA ligase, T7 DNA ligase, and T4 RNA ligase.
  • the ligase of the invention links the linker in the tail-control component to the substrate-tailed dGTP or dCTP polynucleotide.
  • the ligase of the invention links a 5' sequencing linker to a synthetic double stranded deoxy polynucleotide.
  • the polynucleotide substrate is purified. Purification of the polynucleotide substrate is carried out by any method known and understood by those skilled in the art.
  • Purification of the polynucleotide substrate of the present invention can be carried out by adding magnetic beads whose surface is carboxyl modified. In other embodiments, purification of the polynucleotide substrate is carried out by column purification and precipitation.
  • Phos phosphoric acid
  • C3Spacer carbon 3 arm
  • rC cytosine ribonucleotide
  • rG guanine ribonucleotide
  • 5mC 5-methyl-cytosine deoxynucleotide
  • D dA, dT or dG Nucleotide
  • N dA, dT, dC or dG nucleotide
  • Phos phosphoric acid
  • C3 Spacer carbon 3 arm
  • rC cytosine ribonucleotide
  • Phos phosphoric acid
  • C3 Spacer carbon 3 arm
  • rC cytosine ribonucleotide
  • Phos phosphoric acid
  • 5mC 5-methyl-cytosine deoxynucleotide
  • nucleotide homo-tails to a mixed single-stranded DNA polynucleotide substrate having different bases at the 3' end using a TdT enzyme
  • TdT enzyme Enzymatics, catalog number P7070L, 20U/ ⁇ L
  • dATP (Takara, catalog number 4026, 100 mM)
  • dGTP (Takara, catalog number 4027, 100 mM)
  • dTTP (Takara, catalog number 4029, 100 mM)
  • Substrate preparation DNA polynucleotide substrate 001 (3A end dA), 002 (3' end dG), 003 (3' end dT), 004 (3' end dU) and Mixing 005 (dC at the 3' end) in equimolar amounts to obtain a mixed single-stranded DNA polynucleotide substrate having different nucleotides at the 3' end to mimic bisulfite treatment or DNA polynucleotide after damage 3' terminal nucleotide composition.
  • Substrate denaturation The mixed single-stranded DNA polynucleotide substrate was incubated at 95 ° C for 2 minutes and then rapidly placed on ice to maintain the substrate in a single-stranded state.
  • nucleotides to the tail-end (tailing) reaction was carried out in the reaction solution; the reaction solution was incubated at 37 ° C for 5 minutes, 15 minutes and 30 minutes, respectively, and then incubated at 70 ° C for 10 minutes to inactivate the TdT enzyme.
  • Lane 1 is a 20-500 bp DNA marker
  • Lane 2 is a pre-tailed substrate
  • Lanes 3-6, 7-10 and 11-14 are TdT enzymes for substrate addition (dG). ), poly(dC), poly(dA) and poly(dT) tails and reacted for 5 minutes, 15 minutes and 30 minutes of product.
  • the addition of poly(dG) and poly(dC) tails to the TdT enzyme consumed substrate more rapidly, significantly faster than the addition of poly(dA) and poly(dT) tails (lanes 3-14).
  • the TdT enzyme has a higher dissociation constant for the complex formed by adding poly(dG) or poly(dC) tail to the substrate, and the dissociation constant of the complex formed by adding poly(dA) or poly(dT) tail is also lower.
  • the TdT enzyme can be dissociated by adding less poly(dG) or poly(dC) tails after binding to the substrate, so that the enzyme molecule has the opportunity to bind to the next substrate, so the utilization of the substrate is sufficient.
  • the tail product of the substrate was more diffuse, indicating that the length of the four polynucleotides added was uncontrollable (lanes 3-14).
  • TdT enzyme is more fully utilized for the substrate when the poly(dG) and poly(dC) tails are added to the mixed single-stranded DNA polynucleotide substrate with different nucleotides at the 3' end. Under the conditions of Example 1, the tail length of the substrate was not controllable.
  • nucleotide homo-tails to a mixed single-stranded DNA polynucleotide substrate containing a portion of a random sequence and having different nucleotides at the 3' end using a TdT enzyme
  • TdT enzyme Enzymatics, catalog number P7070L, 20U/ ⁇ L
  • TdT enzyme (New England Biolabs, catalog number M0315L, 20U/ ⁇ L)
  • TdT enzyme (ThermoFisher Scientific, catalog number EP0161, 20U/ ⁇ L)
  • Substrate preparation DNA polynucleotide substrate 006 (3A end dA), 007 (3' end dT), 008 (3' end dC), 009 (3' end dG) and 010 (dU at the 3' end) was mixed in equimolar amounts to give a mixed single-stranded DNA polynucleotide substrate having a partially random sequence and a different nucleotide at the 3' end.
  • Reaction Formulation containing 1 pmol of denatured substrate (polynucleotide 006-010 0.25 pmol each), 1x TdT reaction buffer, 200 ⁇ M dGTP or dCTP or dATP or dTTP, and 10 units of Enzymatics or New England Biolabs or ThermoFisher Scientific The 5 ⁇ L reaction solution of the produced TdT enzyme was incubated at 37 ° C for 30 minutes, and then incubated at 70 ° C for 10 minutes to inactivate the TdT enzyme.
  • Lane 1 is a 20-500 bp DNA marker and Lane 2 is a pre-tailed substrate.
  • the TdT enzyme was more fully utilized when adding poly(dG) and poly(dC) tails. This result is consistent with the results of Example 1 (lane 3 - 6), and the TdT enzymes produced by the three manufacturers Enzymatics, New England Biolabs and ThermoFisher Scientific have similar substrate utilization rates (lanes 3-6, 7-10 and 11-14).
  • the substrate residue after the reaction in this example is slightly increased and may be caused by a complex secondary structure formed by a partial random sequence (lane 3).
  • the TdT enzyme has a higher utilization of the substrate when the poly(dG) and poly(dC) tails are added to the mixed single-stranded DNA polynucleotide substrate with different nucleotides at the 3' end. This property is independent of the substrate. Sequence characteristics and producers of TdT enzymes.
  • DNA marker for labeling substrate tail length No. 021, 022 (Table 1)
  • 10x green buffer (Enzymatics, catalog number B0120, 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, pH 7.9)
  • TdT enzyme dGTP, 2x RNA loading buffer and 20-500 bp DNA label (manufacturer and catalog number same as Example 1) method:
  • reaction solution for the tailing reaction 5 ⁇ L of the reaction solution for the tailing reaction was prepared as shown in Table 3-1 below, and the cells were incubated at 25 ° C, 37 ° C, and 45 ° C for 15 minutes, respectively, and then incubated at 70 ° C for 10 minutes to inactivate the TdT enzyme. .
  • Lane 1 is a 20-500 bp DNA marker
  • Lane 2 is a pre-tailing substrate
  • Lane 3 is a reaction product of a TdT enzyme adding a poly(dG) tail to a substrate at 25 ° C
  • 4-13 are the reaction products of the tailing molecule 011-020, respectively, and tailing at 25 ° C
  • lanes 14, 26 and 38 are multi-cores of 60b and 70b for labeling the substrate to add poly (dG) tail length.
  • Glycosidic acid markers (021 and 022).
  • Lane 15 is the reaction product of the TdT enzyme adding a poly(dG) tail to the substrate at 37 ° C; Lanes 16-25 are the reaction products respectively adding a tail-control molecule 011-020 and tailing at 37 ° C; Lane 27 The reaction product of the poly(dG) tail was added to the substrate for the TdT enzyme at 45 ° C; lanes 28-37 were the reaction products of the tail-control molecule 011-020, respectively, and tailing at 45 ° C.
  • the TdT enzyme does not fix the tail length of the substrate at 25 ° C, 37 ° C and 45 ° C, and the tail length is concentrated near 100 b (lanes 3, 15 and 27).
  • the tailing efficiency of the DNA polynucleotide substrate e.g., double-stranded DNA having a 3' recess or blunt end
  • the tail-controlling molecule can anneal to the poly(dG) tail added to the substrate by the TdT enzyme over a certain temperature range, forming a 3' hidden double-stranded structure, thereby reducing the addition.
  • Tail efficiency and limit the length of the tail In the presence of a tail-controlling molecule, a tail-control molecule with 7-20b poly(dC) can control the tail length of the substrate, and the tail-control molecules with 7-20b, 8-20b and 9-20b poly(dC) can The length of the poly(dG) tail added to the substrate by the TdT enzyme was controlled at about 20b (lanes 6-13, 19-24 and 32-37) under the reaction conditions of 25 ° C, 37 ° C and 45 ° C.
  • the controlled polynucleotide added to the polynucleotide substrate by the TdT enzyme is used as the complementary binding region of the extended primer to synthesize the second strand, thereby constructing a methylation sequencing library.
  • dNTP (Takara, catalog number 4030, each 2.5 mM)
  • RNase A (Takara, Cat. No. 2158, 10 mg/mL)
  • a tailing reaction mixture was prepared as shown in Table 4-1, and the mixture was incubated at 37 ° C for 15 minutes, at 95 ° C for 2 minutes, and then at 4 ° C.
  • the ⁇ -DNA was treated with bisulfite, and the degradable tail-tailing polynucleotide (025) was used to make the TdT enzyme add a controllable poly(dG) tail to the treated ⁇ -DNA, and then added
  • the P5 region, the sample tag region and the P7 region (Table 7) of the Illumina Truseq library were amplified by PCR to obtain a structurally complete final sequencing library.
  • the fragment distribution detection showed that the average size of the library fragment was 328 bp, no linker dimer, The library was shown to be of high purity (as shown in Figure 8); qPCR results showed that the library had a molar concentration of 191.3 nM, demonstrating that the bisulfite-treated lambda-DNA was immobilized by the sequencing linker and became an efficient sequencing library.
  • the tail-tailing molecular polynucleotide allows the TdT enzyme to add a controlled polynucleotide homologous tail to the substrate, which can be used as a binding region for the extension primer; after the double-strand synthesis, the "5' sequencing linker" is connected, and PCR amplification is performed. Increased, an efficient sequencing library can be obtained.
  • the tail-controlling molecule is used to control the length of the poly(dG) tail added to the substrate by the TdT enzyme and the poly(dG) tail of the substrate is linked to the linker by the action of the ligase.
  • Short linker polynucleotide No. 024 (Table 1)
  • E. coli DNA ligase (New England Biolabs, catalog number M0205S, 10 U/ ⁇ L)
  • Lane 1 is the substrate before tailing
  • Lane 2 is the reaction product of TdT enzyme adding poly(dG) tail to the substrate at 25 °C
  • Lanes 3-12 are added respectively.
  • Tail link 011/024-020/024 (as shown in Table 2) and the reaction product incubated at 25 ° C
  • Lane 13 is the reaction product of the TdT enzyme adding a poly(dG) tail to the substrate at 37 ° C
  • 14-23 are the reaction products respectively added with a tail-control joint 011/024-020/024 (as shown in Table 2) and incubated at 37 ° C
  • Lane 24 is a TdT enzyme added to the substrate at 45 ° C ( dG)
  • Lanes 25-34 are the reaction products which were respectively added with a tail-control joint 011/024-020/024 (as shown in Table 2) and incubated at 45 °C.
  • the poly(dG) tail added to the substrate by the TdT enzyme is uncontrollable in the absence of the tail-control link (lanes 2, 13 and 24); after addition of the tail-control linker and ligase, the tailing sequence of the substrate is obtained and short
  • the linker (024) is subjected to the ligation product after the ligation reaction (lanes 3-12, 14-23 and 25-34); when reacted at 25 ° C, 37 ° C and 45 ° C, the poly(dC) tail length is different.
  • Controlled tail adaptors (as shown in Table 2) and ligase were able to obtain the product of the substrate attached to the short linker (024) after tailing (as shown in lanes 3-12, 14-23 and 25-34);
  • the tail-control effect of the tail-end joint with poly (dC) tail of 7b or more is better.
  • the TdT enzyme and ligase can complete the hybrid single Poly(dG) controlled tailing reaction of a stranded DNA polynucleotide substrate and ligation of the tail with a linker; control of 5-20b poly(dC) tails when performing the above reaction between 25-45 °C
  • the tail linker can obtain the product of the substrate attached to the linker after tailing.
  • the TdT enzyme adds a poly(dG) tail of indefinite length to the substrate (lane 2), and when the tail-control linker (016/024) is present, the TdT enzyme pair The length of the tail of the substrate is controlled (lane 3).
  • Simultaneous addition of TdT enzyme, tail-controlling linker and E. coli DNA ligase for 1 minute gave a ligation product of a substrate with a fixed length poly(dG) tail and a short linker (024) (lane 4); The amount of ligation product remained unchanged (lanes 5-12), indicating that the tailing of the substrate and the ligation reaction were completed quickly.
  • Non-degradable tail-controlling polynucleotide 016 (Table 1)
  • dNTP (Takara, catalog number 4030, each 2.5 mM)
  • RNase A (Takara, Cat. No. 2158, 10 mg/mL)
  • the bisulfite-treated ⁇ -DNA was denatured into a single-stranded state, and the substrate was added in the presence of TdT enzyme, E. coli DNA ligase, and tail-control link (025/026 or 016/026).
  • the controlled poly(dG) tail is ligated to the long linker; the tail-tailing molecular polynucleotide (025 or 016) is replaced by degradation or stranding and the two-strand synthesis is completed; and the substrate DNA of the double-stranded synthesis is completed with "5'
  • the sequencing linker is ligated and amplified by PCR to obtain a sequencing library with a complete structure. As shown in FIG. 11A and FIG.
  • the distribution of the library fragments obtained by the above two methods is in the range of 150-1000 bp, and there is no linker dimer, and the library has high purity.
  • concentration of the sequencing libraries obtained by the two two-strand synthesis methods were similar, 588.9 nM and 707.4 nM, respectively, which met the sequencing requirements of the Illumina sequencer (the sequencing requirement of the Illumina Nextseq sequencer was: the library volume was greater than or equal to 1.3).
  • the library concentration is greater than or equal to 1.8 nM; the sequencing requirements of the Illumina xTen sequencer are: library volume greater than or equal to 5 ⁇ l, library concentration greater than or equal to 3 nM).
  • ⁇ -DNA bisulfite treatment kit, 5x annealing buffer, 10x green buffer, TdT enzyme, E. coli DNA ligase, dGTP, ⁇ -nicotinamide adenine dinucleotide, 10x isothermal amplification buffer II, dNTP, Bst 3.0 DNA polymerase, RNase A, 2x T4 DNA rapid ligation buffer, T4 DNA ligase, 2x high fidelity hot start methylation PCR mix, Beckman Ampure XP magnetic beads and enzyme-free water ( Manufacturer and catalog number are the same as in the example 7)
  • Methylation sequencing library preparation A methylation sequencing library was constructed according to the method described in Example 7 using 10 ng of ⁇ -DNA, wherein the two-strand synthesis was carried out according to a degradation type method.
  • the experimental results are shown in Figure 12.
  • the methylation sequencing library was constructed using a single-stranded polynucleotide with a stem-loop-structured tail-crossing linker (030).
  • the average size of the final sequencing library fragment was 396 bp, no linker II.
  • the purity of the library was high; the qPCR assay showed that the library concentration was 15.2 nM, indicating that the DNA substrate was immobilized by the sequencing linker, and an efficient sequencing library was obtained.
  • ⁇ -DNA bisulfite treatment kit, 5x annealing buffer, 10x green buffer, TdT enzyme, E. coli DNA ligase, dGTP, ⁇ -nicotinamide adenine dinucleotide, 10x isothermal amplification buffer II, dNTP, Bst 3.0 DNA polymerase, T4 DNA ligase, 2x high fidelity hot start methylation PCR mix, Beckman Ampure XP magnetic beads and enzyme-free water (manufacturer and catalog number same as Example 7)
  • a non-degradable tail joint was prepared according to the joint preparation method of Example 4 (011/026, 012/026, 013/026, 014/026, 015/026, 016/026, 017/026, 018/026, 019/026, and 020/026, as shown in Table 3) and the dt "5' sequencing linker" (031/032, as shown in Table 4).
  • Methylation sequencing library preparation a methylation sequencing library was constructed according to the method described in Example 7 using 6.75 ng of ⁇ -DNA; wherein the tailing and ligation reactions were respectively carried out using a poly(dC) tail length of 5-20b Controlled tail joints (011/026, 012/026, 013/026, 014/026, 015/026, 016/026, 017/026, 018/026, 019/026, and 020/026), two-chain synthesis The chain displacement extension method is carried out.
  • the experimental results are shown in Fig. 13A to Fig. 13J, and the fragmentation of the methylation sequencing library constructed by the poly(dC) tail length of 5-20b to the methylated sequencing library after the bisulfite treatment is distributed in the range of 200-1000 bp.
  • Meshless dimer, high library purity as shown in Table 9-1, the lowest concentration of the library constructed using the 5b poly(dC) tailed tailer was 6.2 nM with a tail of 20b poly(dC) tail
  • the library constructed by the linker had the highest concentration of 31.7 nM.
  • the tail-tailing linker with 5-20b poly(dC) tail can effectively construct methylation sequencing library of bisulfite-treated DNA.
  • 10x End Repair Buffer (New England Biolabs, Cat. No. B6052S, 50 mM Tris-HCl, 10 mM Magnesium Chloride, 10 mM Dithiothreitol, 1 mM Adenosine Triphosphate, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP, pH 7.5 )
  • T4 DNA polymerase Enzymatics, catalog number P7080L, 3U/ ⁇ L
  • 10x dA tailing buffer (New England Biolabs, Cat. No. B6059S, 10 mM Tris-HCl, 10 mM magnesium chloride, 50 mM sodium chloride, 1 mM dithiothreitol, 0.2 mM dATP, pH 7.9)
  • Bisulfite treatment kit 5x annealing buffer, 10x green buffer, TdT enzyme, E. coli DNA ligase, dGTP, ⁇ -nicotinamide adenine dinucleotide, 10x isothermal amplification buffer II, dNTP, Bst 3.0 DNA Polymerase, T4 DNA Rapid Link Buffer, T4 DNA Fast Ligase, 2x High Fidelity Hot Start Methylation PCR Mix, Beckman Ampure XP Magnetic Beads and Enzyme Free Water (Manufacturer and Catalog Number Same as Example 7 )
  • the terminal repair reaction mixture was prepared as shown in Table 10-1, and reacted at 20 ° C for 30 minutes, and then 45 ⁇ l of Beckman Ampure XP magnetic beads were added to recover the repaired DNA, and eluted with 26 ⁇ l of enzyme-free water.
  • PCR amplification reaction mixture was prepared, except that the P7 PCR tag primer was a polynucleotide numbered 033; followed by the PCR amplification procedure shown in Table 4-5. The procedure was carried out except that the number of amplification cycles was 18; after the reaction was over, the PCR product was recovered using 75 ⁇ l of Beckman Ampure XP magnetic beads and eluted with 20 ⁇ l of enzyme-free water to obtain a final sequencing library.
  • the experimental results are shown in Table 10-4 below.
  • the concentration of the methylation sequencing library constructed from 10 ng of human genomic DNA based on the TdT enzyme and the tail-tailing method was 151.8 nM, while the library concentration of the conventional method was 99.6 nM.
  • the amount of sequencing data is 19.4Gb
  • the library constructed by single-strand tail-joining method has a redundancy of 13.4%, covering 89.6% of CpG regions, and the average sequencing depth is 4.8x.
  • the library sequencing data constructed by the traditional method is 22.0.
  • the redundancy of Gb reached 70.4%, covering only 13.8% of CpG region, and the average sequencing depth was 0.3x.
  • the tail-joining method based on TdT enzyme and tail-tailing can efficiently construct methylation sequencing library of a small amount of human genomic DNA.
  • the efficiency of database construction and sequencing data are far superior to traditional methods.

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Abstract

L'invention concerne un procédé de construction de bibliothèque de séquençage à haut rendement de seconde génération, et un kit. La présente invention améliore le taux d'utilisation d'un modèle d'acide nucléique, simplifie la procédure de construction d'une bibliothèque de séquençage et rend un résultat de séquençage plus précis et le degré de couverture plus uniforme.
PCT/CN2017/109770 2017-11-07 2017-11-07 Procédé de construction de bibliothèque de séquençage à haut rendement de seconde génération WO2019090482A1 (fr)

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