CN110607353B - Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology - Google Patents
Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology Download PDFInfo
- Publication number
- CN110607353B CN110607353B CN201910808734.5A CN201910808734A CN110607353B CN 110607353 B CN110607353 B CN 110607353B CN 201910808734 A CN201910808734 A CN 201910808734A CN 110607353 B CN110607353 B CN 110607353B
- Authority
- CN
- China
- Prior art keywords
- adapter
- chain
- dna
- truncated
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method and a kit for rapidly preparing a DNA sequencing library by utilizing an efficient ligation technology, wherein the method comprises the following steps: DNA3 'linker ligation, synthesis of complementary strand, DNA5' linker ligation, and library amplification. The kit comprises the following components: a tool enzyme, a tool enzyme reaction system, a linker system, an extension system, a PCR reaction system and Low-EDTA-TE. The method realizes the successful library construction of various trace DNA samples, breaks through the conventional library construction means, avoids the DNA loss caused by end repair, and reflects the real information of the samples as much as possible.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a technology for efficiently connecting sequencing joints at the tail ends of DNA (deoxyribonucleic acid) in sequence to realize library construction of various complex samples and trace DNA.
Background
AT present, the conventional library construction strategy is to repair the ends of fragmented DNA, then to connect sequencing adapters through AT, and to amplify the library by PCR. However, the conventional database building requires high quality of sample DNA, large input amount, and can not realize normal database building of samples with serious damage, especially samples mainly existing in single-stranded DNA. And when the input amount is too low, the conventional library building cannot be finished.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method and a kit for rapidly preparing a DNA sequencing library by utilizing an efficient connection technology, so that successful library construction of various trace DNA samples is realized, the conventional library construction means is broken, the DNA loss caused by terminal repair is avoided, and the real information of the samples is reflected as much as possible.
The invention relates to a method for rapidly preparing a DNA sequencing library by utilizing an efficient ligation technology, which is characterized by comprising the following steps of:
s1, DNA3' joint connection: firstly, adding a section of polynucleotide PolyA or PolyC or PolyG or PolyT to the 3 'end of DNA to be detected by using terminal transferase to obtain a DNA-polynucleotide structure, finishing the connection of the 3' end of the DNA-polynucleotide and a T7Truncated Adapter joint by using DNA repair enzyme, DNA polymerase and DNA ligase, and synthesizing a DNA-polynucleotide-T7 Truncated Adapter;
the DNA to be detected in the step S1 is a single-stranded DNA fragment or a double-stranded DNA fragment, or comprises the single-stranded DNA fragment and the double-stranded DNA fragment at the same time, when the DNA to be detected comprises the double-stranded DNA, the step S1 also comprises a denaturation step, and the denaturation step denatures the double-stranded DNA into the single-stranded DNA;
s2, synthesis of a complementary strand: synthesizing a complementary strand of the DNA-poly oligonucleotide-T7 Truncated Adapter single strand added in the step S2 by using DNA polymerase to synthesize double-stranded DNA;
s3, DNA5' joint connection: connecting a T5 Truncated Adapter adaptor to the 5' end of the DNA double strand in the step S3 by using DNA ligase through a blunt end or AT connection mode;
s4, library amplification: amplifying a PCR library by using a DNA polymerase reaction solution to obtain a library sample capable of being subjected to on-machine sequencing;
the T7Truncated Adapter consists of a first chain of the T7Truncated Adapter and a second chain of the T7Truncated Adapter, wherein the second chain of the T7Truncated Adapter is a chain 2a of the T7Truncated Adapter, a chain 2b of the T7Truncated Adapter, a chain 2c of the T7Truncated Adapter or a chain 2d of the T7Truncated Adapter, a5' end region of the first chain of the T7Truncated Adapter and a5' end region of the second chain of the T7Truncated Adapter are complementary into double-stranded DNA, a3' end of the second chain of the T7Truncated Adapter is an oligonucleotide selected from PolyT, polyA, polyG or PolyC; the 5 'end of the first chain of the T7Truncated Adapter is subjected to phosphate modification, the 3' end of the first chain of the T7Truncated Adapter is subjected to blocking modification, the 5 'end and the 3' end of the second chain of the T7Truncated Adapter are subjected to blocking modification, and the blocking modification group is selected from amino, C3Space, C6 Space or biotin;
the nucleotide sequence number of the first chain of the T7Truncated Adapter is shown as SEQ ID No.1, the nucleotide sequence number of the 2a chain of the T7Truncated Adapter is shown as SEQ ID No.2, the nucleotide sequence number of the 2b chain of the T7Truncated Adapter is shown as SEQ ID No.3, the nucleotide sequence number of the 2c chain of the T7Truncated Adapter is shown as SEQ ID No.4, and the nucleotide sequence number of the 2d chain of the T7Truncated Adapter is shown as SEQ ID No. 5;
the T5 Truncated Adapter consists of a first chain of the T5 Truncated Adapter and a second chain of the T5 Truncated Adapter, wherein a3' end region of the first chain of the Truncated Adapter and a5' end region of the second chain of the T5 Truncated Adapter are complemented into double-stranded DNA, and a5' end region of the first chain of the T5 Truncated Adapter exists in a single-stranded form; the 5 'end of the first chain of the T5 Truncated Adapter is subjected to blocking modification, the 3' end of the first chain of the T5 Truncated Adapter is subjected to thio modification, the 5 'end and the 3' end of the second chain of the T5 Truncated Adapter are subjected to blocking modification, the blocking modification group is selected from amino, C3 Spacer, C6 Spacer or biotin, and the first chain of the T5 Truncated Adapter is a T5 Truncated Adapter chain 1a or a T5 Truncated Adapter chain 1b;
the nucleotide sequence number of the T5 Truncated Adapter chain 1a is shown in SEQ ID No.6, the nucleotide sequence number of the T5 Truncated Adapter chain 1b is shown in SEQ ID No.7, and the nucleotide sequence number of the T5 Truncated Adapter chain second is shown in SEQ ID No. 8.
Further, the DNA ligase is selected from T4 DNA ligase, taq DNA ligase, e.
Still further, the DNA polymerase is selected from Taq polymerase, A family DNA polymerase, B family high fidelity DNA polymerase.
Further, the DNA repair enzyme is selected from the group consisting of T4PNK Klenow, FEN1 and Endonuclease V.
Still further, the linker system of the T7Truncated adapter is a combinatorial enzyme selected from the group consisting of TDT, e.coli DNA Ligase, T4PNK, klenow, FEN1 and Endonuclease V.
Furthermore, the DNA to be tested in step S1 is one of genome gDNA, FFPE DNA, cell free DNA, chIP DNA, archaea, virus DNA, RNA-seq single-stranded cDNA and Bisiute DNA.
The invention also provides a kit for rapidly preparing the DNA sequencing library by using the method and utilizing an efficient ligation technology, which is characterized by comprising the following components:
(1) Tool enzyme: the tool enzyme comprises terminal transferase, DNA repair enzyme, DNA polymerase and DNA ligase;
(2) Tool enzyme reaction system: buffer solution required by the reaction corresponding to each tool enzyme;
(3) A linker system: t7Truncated Adapter joint and T5 Truncated Adapter joint;
(4) An extension system: a T7 primer;
(5) A PCR reaction system;
(6)Low-EDTA-TE；
the T7Truncated Adapter and the T5 Truncated Adapter are linkers containing special modified bases;
the specific modified base includes a base for increasing the ligation efficiency at the 5 'end and preventing chain extension or a base for preventing OH extension at the 3' end of the target DNA library; the special modified basic group is phosphate group or amino, C3 Spacer, C6 Spacer, biotin and the like.
Further, the T7Truncated Adapter joint is designed as shown in FIG. 1;
the T7Truncated Adapter consists of a first chain of the T7Truncated Adapter and a second chain of the T7Truncated Adapter, wherein the second chain of the T7Truncated Adapter is a chain 2a of the T7Truncated Adapter, a chain 2b of the T7Truncated Adapter, a chain 2c of the T7Truncated Adapter or a chain 2d of the T7Truncated Adapter, a5' end region of the first chain of the T7Truncated Adapter and a5' end region of the second chain of the T7Truncated Adapter are complementary into double-stranded DNA, a3' end of the second chain of the T7Truncated Adapter is an oligonucleotide selected from PolyT, polyA, polyG or PolyC; the 5 'end of the first chain of the T7Truncated Adapter is subjected to phosphate modification, the 3' end of the first chain of the T7Truncated Adapter is subjected to blocking modification, the 5 'end and the 3' end of the second chain of the T7Truncated Adapter are subjected to blocking modification, and the blocking modification group is selected from amino, C3Space, C6 Space or biotin;
the nucleotide sequence number of the first chain of the T7Truncated Adapter is shown as SEQ ID No.1, the nucleotide sequence number of the 2a chain of the T7Truncated Adapter is shown as SEQ ID No.2, the nucleotide sequence number of the 2b chain of the T7Truncated Adapter is shown as SEQ ID No.3, the nucleotide sequence number of the 2c chain of the T7Truncated Adapter is shown as SEQ ID No.4, and the nucleotide sequence number of the 2d chain of the T7Truncated Adapter is shown as SEQ ID No. 5;
the T5 Truncated Adapter consists of a first chain of the T5 Truncated Adapter and a second chain of the T5 Truncated Adapter, wherein a3' end region of the first chain of the Truncated Adapter and a5' end region of the second chain of the T5 Truncated Adapter are complemented into double-stranded DNA, and a5' end region of the first chain of the T5 Truncated Adapter exists in a single-stranded form; the 5 'end of the first chain of the T5 Truncated Adapter is subjected to blocking modification, the 3' end of the first chain of the T5 Truncated Adapter is subjected to thio modification, the 5 'end and the 3' end of the second chain of the T5 Truncated Adapter are subjected to blocking modification, the blocking modification group is selected from amino, C3 Spacer, C6 Spacer or biotin, and the first chain of the T5 Truncated Adapter is a T5 Truncated Adapter chain 1a or a T5 Truncated Adapter chain 1b;
the nucleotide sequence number of the T5 Truncated Adapter chain 1a is shown in SEQ ID No.6, the nucleotide sequence number of the T5 Truncated Adapter chain 1b is shown in SEQ ID No.7, and the nucleotide sequence number of the T5 Truncated Adapter chain second is shown in SEQ ID No. 8.
Still further, the DNA repair enzyme is selected from T4PNK, klenow, FEN1 and endonucleoclean V;
the DNA polymerase is selected from Taq polymerase, A family DNA polymerase and B family high-fidelity DNA polymerase;
the DNA ligase is selected from T4 DNA ligase, taq DNA ligase and E.
Still further, the linker system of the T7Truncated Adapter is a combinatorial enzyme selected from the group consisting of TDT, e.coli DNA Ligase, T4PNK, klenow, FEN1 and endonucleoase V.
The beneficial effects of the invention are as follows: the invention realizes the successful bank building of various DNA samples, breaks through the conventional bank building means, avoids the loss of DNA caused by end repair and reflects the real information of the samples as much as possible. The invention has great advantages compared with the prior art especially when establishing a library aiming at the trace DNA sample of 5 pg-1 mu g: 1. the invention has no selectivity on DNA samples, and the DNA to be detected can be double-stranded DNA, single-stranded DNA, complete DNA or damaged DNA fragments; 2. the invention can be used for constructing a library aiming at trace DNA samples, and the weight of the samples can be as low as 5 pg-1 mug; 3. efficient DNA library construction can be tested for simple samples or complex samples.
Drawings
FIG. 1 is a schematic diagram of a T7Truncated Adapter structure using the method of the present invention;
FIG. 2 is a schematic diagram of a T5 Truncated Adapter structure using the method of the present invention;
FIG. 3 is a schematic representation of the efficiency analysis of the addition of PolyA at the end of oligonucleotide N1 using the method of the invention;
FIG. 4 is a schematic diagram of the analysis of efficiency of terminal transferase with PolyA at different incubation times in a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention;
FIG. 5 is a schematic diagram of the analysis of 3' linker efficiency of a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention;
FIG. 6 is a schematic diagram of the analysis of 3' linker efficiency in a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention;
FIG. 7 is a schematic diagram of the analysis of complementary strand synthesis in a method for rapid preparation of a DNA sequencing library by using efficient ligation;
FIG. 8 is a schematic diagram of 5' linker efficiency analysis of a method for rapid preparation of DNA sequencing libraries using efficient ligation;
FIG. 9 is a schematic diagram of PCR amplification efficiency analysis of a method for rapidly preparing a DNA sequencing library by using a high-efficiency ligation technique according to the present invention;
FIG. 10 is a schematic diagram of library preparation of different samples of a method for rapid preparation of DNA sequencing libraries using efficient ligation techniques according to the present invention;
FIG. 11 is a library preparation diagram of trace samples of a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings in conjunction with the following detailed description. It should be understood that the description is intended to be exemplary only, and is not intended to limit the scope of the present invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
Example 1
1.1 Addition of DNA-terminal PolyA:
in this example, the addition of PolyA at the N1' end was analyzed using N1 (Shanghai Czeri Bio Inc.) oligonucleotides as the study substrate, the N1 nucleotide sequence being shown in SEQ ID No.9, using Terminal Transferase (Terminal Transferase, tdT, new England Biolabs/NEB, M0315):
1.2 Specific PolyA addition step:
the reaction system is prepared as follows:
and (3) running a reaction program: 37 ℃/15min,72 ℃/10min,4 ℃/hold.
1.3 Test results):
by comparing different 10 XTdT Buffer components, the following table shows that different reaction buffers have different tail-adding abilities to the oligonucleotide ends, as shown in FIG. 3, wherein No.1 is a negative control N1, no. 2-6 are respectively 10 XTdT buffers 1-5, and 10 XTdT buffers 5 adding PolyA has the best effect, and can be added to more than 10 PolyA. The 10 XTdT Buffer 5 shows basically consistent performance at different incubation times at 37 ℃, the difference of the added PolyA numbers is small, as shown in figure 4, wherein No.1 is negative control N1, no. 2-4 are respectively 10 XTdT Buffer 5, and the incubation is respectively carried out at 37 ℃ for 15min, 20min and 30min.
Example 2
2.1 DNA3' linker ligation efficiency test:
this example used random amplifications of-150 bp (S1) and-300 bp (S2) from plasmid pBC4 (NEB, N0354) as DNA substrates for 3' linker efficiency testing. The nucleotide sequences of the primers for amplifying the S1 are shown as SEQ ID No.10 and 11. The nucleotide sequence of the primer for amplifying the S2 is shown as SEQ ID No.12 and 13. Reagents 10 XTdT Buffer 5, commercial 10U TdT (NEB, M0315), 60U E.coli DNA ligase (NEB, M0205), 10U T4PNK (T4 Polynucleotide Kinase, NEB, M0201) were used, wherein TdT could complete the addition of mononucleotide at the 3' end of DNA, E.coli DNA ligase could complete the ligation of PolyA and the Nick of T7 linker, and T4PNK could repair the 5' end of DNA, facilitating ligation of 5' linker. Klenow (NEB, M0212), pol1 (NEB, M0210), FEN1 (NEB, M0645) and Endonuclease V (NEB, M0305) with 3' -5' exonuclease activity, which complements or cleaves away excess PolyA, were tested for their effect on the efficiency of 3' linker ligation. This procedure completes the ligation via PolyA and the T7 truncatadedapter linker. The T7Truncated Adapter linker structure is shown in FIG. 1, and the nucleotide sequence is: the sequence number of the first strand of nucleotide is shown as SEQ ID No.1, and the sequence number of the second strand of nucleotide is shown as SEQ ID No. 2. The 5 'end of the T7 joint is adenosine modified by phosphate groups, the 3' end of the T7 joint is cytosine modified by amino groups, and the chemical modification of the T7 joint can inhibit nonspecific extension and increase the connection efficiency with the PolyA.
The method can realize the connection process of adding PolyA and T7Truncated Adapter joints at the 3' end of the DNA with different substrates pre-denatured into single chains.
2.2 Experimental procedure):
the reaction system is prepared as follows:
the reaction was carried out at 37 ℃ for 15 minutes and at 95 ℃ for 2 minutes of inactivation.
1.8 × AMPure XP magnetic beads are fully recovered.
2.3 Results of the experiment)
As shown in FIG. 5, A is the S1 negative control, B is the experimental group 1, C is the experimental group 2, and D is the experimental group 3.
As shown in FIG. 6, where A is the S2 negative control, B is experimental group 1, C is experimental group 2, and D is experimental group 3.
As can be seen, klenow, FEN1 and IndoV all can achieve a3' ligation efficiency of S1/S2 of 90% or more.
Example 3
3.1 ) complementary strand synthesis:
complementary strand synthesis was continued using-150 bp (S1) and-300 bp (S2) randomly amplified from plasmid pBC4 (NEB, N0354) as DNA substrates. The efficiency of complementary strand synthesis was tested using a commercial reagent 2 XKAPA HiFi HotStart ReadyMix (Kapa Biosystems/Kapa, KM 2618). This process completes the extension of the complementary strand of denatured single-stranded DNA. The nucleotide sequence number of the T7 primer is shown as SEQ ID No. 14.
The method can realize the synthesis process of the complementary strand of the DNA of the tailing single strands of different substrates.
3.2 Experimental procedure):
the reaction system is prepared as follows:
| component reagent | ||
| DNA3' | 40μL | |
| 2×KAPA HiFi HotStart ReadyMix | 44μL | |
| T7 primer 25. Mu.M | 4μL | |
| General System | 88μL |
The reaction conditions were as follows:
| reaction temperature | Reaction time | |
| 98℃ | 45s | |
| 63℃ | 30s | |
| 68 | 5min | |
| 4℃ | -- |
After the reaction is finished, capturing 1.2 multiplied by AMPure XP magnetic beads, and eluting by using 10 mu L of Low-EDTA-TE;
3.3 Results of the experiment: as shown in FIG. 7, where A is the S1 negative control and B is the extension of S1, it can be seen that S1 can be fully extended using this method.
Example 4
4.1 DNA5' linker ligation efficiency test:
the DNA substrates of-150 bp (S1) and-300 bp (S2) from random amplification of plasmid pBC4 (NEB, N0354) were used successively for the 5' adaptor ligation efficiency test. The reagents used were T4 DNA Ligase (NEB, M0202), T5 linker and 2 XQuick ligation buffer (132 mM Tris-HCl, 20mM MgCl) 2 2mM DTT, 2mM ATP and 15% PEG), DNA5' linker ligation efficiency was tested. This procedure completes the ligation of the T5 Truncated Adapter linker after DNA extension is complete. The T5 Truncated Adapter linker structure is shown in FIG. 2. The nucleotide sequence of the T5 Truncated Adapter linker is: the sequence number of the first strand nucleotide is shown as SEQ ID No.6, and the sequence number of the first strand nucleotide is shown as SEQ ID No. 8. Wherein, the 5' end of the T5 Truncated Adapter linker is modified, so that the connection efficiency can be increased, and the generation of a linker dimer can be reduced. The 5 'end of the T5 Truncated Adapter joint is amino-modified adenine, and the 3' end of the T5 joint is thiothymine.
4.2 Experimental procedure):
the reaction system is prepared as follows:
the reaction conditions were as follows:
| reaction temperature | Reaction time | |
| 25 | 15min | |
| 4℃ | -- |
After the reaction is finished, capturing 1.0 multiplied by AMPure XP magnetic beads, and eluting by using 21 mu L of Low-EDTA-TE;
4.3 Results of the experiment: as shown in FIG. 8, where A is the negative control for S1 and B is the ligation of the 5 'linker of S1, it can be seen that S1 can be ligated at the 5' linker using this method.
Example 5
5.1 Library amplification efficiency test:
PCR amplification efficiency tests were continued using-150 bp (S1) and-300 bp (S2) from random amplification of plasmid pBC4 (NEB, N0354) as DNA substrates. Amplification by PCR was performed using a commercial reagent 2 XKAPA HiFi HotStart ReadyMix (Kapa, KM 2618). The amplification primers were Universal PCR primers (Universal PCR Primer, RM20248, abclonal) and Index primers (Index, RM22201, abclonal).
The method can realize the PCR amplification process of different substrate DNAs.
5.2 Experimental procedure):
the reaction system is prepared as follows:
| component reagent | ||
| Purified T5 | 20μL | |
| 2×KAPA HiFi HotStart ReadyMix | 25μL | |
| Universal PCR primer | 2.5μL | |
| Index primer | 2.5μL | |
| General System | 50μL |
The reaction conditions were as follows
| Reaction temperature | Reaction time | |
| 98℃ | 45s | |
| 98 | 15s | |
| 60℃ | 30s | |
| 72℃ | 30s | |
| 72 | 1min | |
| 4℃ | -- |
After 6 circulation reactions, capturing 1.0 multiplied by AMPure XP magnetic beads, and eluting by using 31 mu L of Low-EDTA-TE;
the experimental results are as follows: as shown in FIG. 9, in which A is PCR amplification of S1 and B is PCR amplification of S2, it can be seen that this method can perform PCR amplification on S1/S2.
Example 6
Testing of different sample library preparations:
in this embodiment, the ligation technique for preparing a DNA library is used to perform a library-building test on common DNA samples from different sources. We tested four samples: comprises animal and plant genome, FFPE DNA, cfDNA and bisufute DNA. The following is a basic DNA library construction procedure, with two replicates per sample set up for testing reliability.
6.1 95 ℃ pretreatment of DNA samples
DNA samples were dosed according to the following table, mixed well, incubated at 95 ℃ for 2min, and immediately placed on ice to fully denature double stranded DNA into single stranded DNA.
6.2 DNA3' linker ligation
A reaction system was prepared as follows, and 10U TdT,60U E.coli DNA ligase,10U T4PNK and 10U Klenow were used in combination as an Enzyme mix.
| Components | Volume of | |
| Pre-denatured DNA | 15μL | |
| T7 joint | 2μL | |
| dATP | 4μL | |
| | 3μL | |
| 10× |
4μL | |
| Adding water to 40 μ L | 40μL |
And (3) running a reaction program: 37 ℃/15min,95 ℃/2min,4 ℃/hold.
6.3 ) Synthesis of complementary strands
The reaction system is prepared as follows:
and (3) running a reaction program: 98 ℃/45s,63 ℃/30s,68 ℃/5min,4 ℃/hold.
6.4 ) purification after complementary strand synthesis
DNA was purified according to the 1.2X AMPure XP magnetic bead ratio. The beads were eluted at 21. Mu.L. mu.L of DNA was taken for the subsequent 5' linker ligation reaction.
6.5 5' linker ligation reaction
The reaction system is prepared as follows:
| component reagent | ||
| Purification of | 20μL | |
| 2×Quick ligation buffer | 25μL | |
| T5 joint | 3μL | |
| T4 DNA Ligase | 2μL | |
| Total volume | 50μL |
And (3) running a reaction program: 25 ℃/15min,4 ℃/hold.
6.6 Purification after 5' linker ligation
DNA was purified according to the 1.0 XAMPure XP magnetic bead ratio. The beads were eluted at 21. Mu.L. 20 μ L of ligated DNA was used for subsequent PCR reactions.
6.7 Amplification of libraries
The reaction system is prepared as follows:
and (3) running a reaction program: 98 ℃/45s;7/9/13/8cycles (98 ℃/10s,60 ℃/30s,72 ℃/30 s); 72 ℃/1min;4 ℃/hold.
8. Purification after library amplification
PCR clean of DNA was performed using 1.0 × AMPure XP DNA purification magnetic beads. The beads were eluted with 31. Mu.L of water. Take 30. Mu.L of DNA library.
The quant was quantified and the yields were as follows:
one of each sample was randomly taken for 2100 analyses, as shown in fig. 10, where a is 200bp fragmented Human gDNA, B is a sequencing library of 200bp fragmented Human gDNA, C is 150bp fragmented FFPE DNA, D is a sequencing library of 150bp fragmented FFPE DNA, E is cfDNA, F is a sequencing library of cfDNA, G is 150bp fragmented bisufute DNA, and H is a sequencing library of 150bp fragmented bisufute DNA, the library size was appropriate and met Illumina sequencing standards.
Example 7
7.1 Test for trace sample library preparation:
in this example, the trace amount of DNA samples were subjected to library construction test by using the ligation technique for DNA library preparation described above. We tested a library of 100pg and 10pg inputs. Referring to example 6, 17 PCR library amplifications were performed on 100pg sample DNA and 18 PCR library amplifications were performed on 10pg sample DNA. The library yields are shown in the following table:
one sample is randomly taken from each sample for 2100 analysis, and as shown in FIG. 11, A is a 200bp fragmented Human gDNA 5pg library, B is a 200bp fragmented Human gDNA 10pg library, and C is a 200bp fragmented Human gDNA 100pg library, and the libraries are appropriate in size and meet Illumina sequencing standards.
It should be understood that the above-described embodiments of the present invention are merely illustrative of or explaining the principles of the invention and are not to be construed as limiting the invention. Therefore, any modification, equivalent replacement, improvement and the like made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. Further, it is intended that the appended claims cover all such variations and modifications as fall within the scope and boundaries of the appended claims or the equivalents of such scope and boundaries.
Sequence listing
<110> Shanghai Engyi Biotech Co., ltd
<120> a method and kit for rapidly preparing a DNA sequencing library using an efficient ligation technique
<130> 201908
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213> T7 Truncated Adapter 1
<400> 1
agatcggaag agcacacgtc tgaactccag t 31
<210> 2
<211> 22
<212> DNA
<213> T7 Truncated Adapter 2a
<400> 2
tgctcttccg atcttttttt tt 22
<210> 3
<211> 22
<212> DNA
<213> T7 Truncated Adapter 2b
<400> 3
tgctcttccg atctaaaaaa aa 22
<210> 4
<211> 22
<212> DNA
<213> T7 Truncated Adapter 2c
<400> 4
tgctcttccg atctgggggg gg 22
<210> 5
<211> 22
<212> DNA
<213> T7 Truncated Adapter 2d
<400> 5
tgctcttccg atctcccccc cc 22
<210> 6
<211> 32
<212> DNA
<213> T5 Truncated Adapter 1a
<400> 6
ctgactttcc ctacacgacg ctcttccgat ct 32
<210> 7
<211> 33
<212> DNA
<213> T5 Truncated Adapter 1b
<400> 7
ctgactttcc ctacacgacg ctcttccgat ctt 33
<210> 8
<211> 13
<212> DNA
<213> T5 Truncated Adapter 2
<400> 8
agatcggaag agc 13
<210> 9
<211> 43
<212> DNA
<213> N1
<400> 9
ctgaggagaa gtctgccgtt actgccctgt ggggctaggt gaa 43
<210> 10
<211> 24
<212> DNA
<213> S1primer F
<400> 10
cagagcagat tgtactgaga gtgc 24
<210> 11
<211> 21
<212> DNA
<213> S1primer R
<400> 11
agctggcgta atagcgaaga g 21
<210> 12
<211> 20
<212> DNA
<213> S2 primer F
<400> 12
<210> 13
<211> 21
<212> DNA
<213> S2 primer R
<400> 13
ctgcgtggaa ctttcgatcc c 21
<210> 14
<211> 20
<212> DNA
<213> T7 primer
<400> 14
Claims (2)
1. A method for rapidly preparing a DNA sequencing library by using an efficient ligation technology is characterized by comprising the following steps:
s1, DNA3' joint connection: firstly, adding a section of polynucleotide to the 3 'end of DNA to be detected by using terminal transferase, wherein the polynucleotide is selected from PolyA, polyC, polyG or PolyT to obtain a DNA-polynucleotide structure, finishing the connection of the 3' end of the DNA-polynucleotide and a T7Truncated Adapter joint by using DNA repair enzyme, DNA polymerase and DNA ligase, and synthesizing the DNA-polynucleotide-T7 Truncated Adapter;
the DNA to be detected in the step S1 is a single-stranded DNA fragment or a double-stranded DNA fragment, or comprises the single-stranded DNA fragment and the double-stranded DNA fragment simultaneously, when the DNA to be detected comprises the double-stranded DNA, the step S1 also comprises a denaturation step, and the denaturation step denatures the double-stranded DNA into the single-stranded DNA;
the T7Truncated Adapter connecting system is a combined enzyme, and the combined enzyme is TdT 10U, E.coli DNA Ligase 60U, T4PNK 10U and Klenow 10U; or TdT 10U, E.coli DNA Ligase 60U, T4PNK 10U and FEN1 5U; or TdT 10U, E.coli DNA Ligase 60U, T4PNK 10U and Endonuclease V5U;
the reaction buffer solution adopted by the T7 ligated Adapter comprises the following components: tris-HCl 700mM, mg (Ac) 2 100mM、NAD + 260uM, DTT 10mM, ATP 10mM, naCl 500mM and KAc 500mM;
s2, synthesis of a complementary strand: synthesizing a complementary strand of the single strand added with the DNA-poly oligonucleotide-T7 Truncated Adapter in the step S1 by using DNA polymerase to synthesize double-stranded DNA;
s3, DNA5' joint connection: connecting a T5 Truncated Adapter adaptor to the 5' end of the DNA double strand in the step S2 by using DNA ligase through a blunt end or AT connection mode;
s4, library amplification: amplifying a PCR library by using a DNA polymerase reaction solution to obtain a library sample which can be subjected to on-machine sequencing;
the T7Truncated Adapter consists of a first chain of T7Truncated Adapter and a second chain of T7Truncated Adapter, wherein the second chain of T7Truncated Adapter is T7Truncated Adapter chain 2a, T7Truncated Adapter chain 2b, T7Truncated Adapter chain 2c or T7Truncated Adapter chain 2d, a5' end region of the first chain of T7Truncated Adapter and a5' end region of the second chain of T7Truncated Adapter are complementary to form a double-stranded DNA, a3' end of the second chain of T7Truncated Adapter is an oligonucleotide selected from PolyT, polyA, polyG or PolyC; the 5 'end of the first chain of the T7Truncated Adapter is subjected to phosphate modification, the 3' end of the first chain of the T7Truncated Adapter is subjected to blocking modification, the 5 'end and the 3' end of the second chain of the T7Truncated Adapter are subjected to blocking modification, and a blocking modification group is selected from amino, C3Space, C6 Space or biotin;
the nucleotide sequence number of the first chain of the T7Truncated Adapter is shown as SEQ ID No.1, the nucleotide sequence number of the 2a chain of the T7Truncated Adapter is shown as SEQ ID No.2, the nucleotide sequence number of the 2b chain of the T7Truncated Adapter is shown as SEQ ID No.3, the nucleotide sequence number of the 2c chain of the T7Truncated Adapter is shown as SEQ ID No.4, and the nucleotide sequence number of the 2d chain of the T7Truncated Adapter is shown as SEQ ID No. 5;
the T5 Truncated Adapter consists of a first chain of the T5 Truncated Adapter and a second chain of the T5 Truncated Adapter, wherein a3' end region of the first chain of the Truncated Adapter and a5' end region of the second chain of the T5 Truncated Adapter are complemented into double-stranded DNA, and a5' end region of the first chain of the T5 Truncated Adapter exists in a single-stranded form; the 5 'end of the first chain of the T5 Truncated Adapter is subjected to blocking modification, the 3' end of the first chain of the T5 Truncated Adapter is subjected to thio modification, the 5 'end and the 3' end of the second chain of the T5 Truncated Adapter are subjected to blocking modification, a blocking modification group is selected from amino, C3 Spacer, C6 Spacer or biotin, and the first chain of the T5 Truncated Adapter is a T5 Truncated Adapter chain 1a or a T5 Truncated Adapter chain 1b;
the nucleotide sequence number of the T5 Truncated Adapter chain 1a is shown in SEQ ID No.6, the nucleotide sequence number of the T5 Truncated Adapter chain 1b is shown in SEQ ID No.7, and the nucleotide sequence number of the T5 Truncated Adapter chain second is shown in SEQ ID No. 8.
2. A kit for rapidly preparing a DNA sequencing library by using a high efficiency ligation technique according to the method of claim 1, which comprises the following components:
(1) Tool enzyme: the tool enzymes are TdT 10U, E.coli DNA Ligase 60U, T4PNK 10U and Klenow 10U; or TdT 10U, E.coli DNA Ligase 60U, T4PNK 10U and FEN1 5U; or TdT 10U, E.coli DNA Ligase 60U, T4PNK 10U and Endonuclase V5U;
(2) Tool enzyme reaction system: buffer solution required by the reaction corresponding to each tool enzyme;
the reaction buffer solution adopted by the T7 ligated Adapter comprises the following components: tris-HCl 700mM, mg (Ac) 2 100mM、NAD + 260uM, DTT 10mM, ATP 10mM, naCl 500mM and KAc 500mM;
(3) A linker system: a T7Truncated Adapter as defined in claim 1 and a T5 Truncated Adapter as defined in claim 1;
(4) An extension system: a T7 primer;
(5) A PCR reaction system;
(6)Low-EDTA-TE。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910808734.5A CN110607353B (en) | 2019-08-29 | 2019-08-29 | Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910808734.5A CN110607353B (en) | 2019-08-29 | 2019-08-29 | Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110607353A CN110607353A (en) | 2019-12-24 |
| CN110607353B true CN110607353B (en) | 2022-11-22 |
Family
ID=68891021
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910808734.5A Active CN110607353B (en) | 2019-08-29 | 2019-08-29 | Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110607353B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20230167490A1 (en) * | 2020-04-28 | 2023-06-01 | Cepheid | Pseudo-complementary bases in genotyping and nucleic acid sequencing |
| CN112176420A (en) * | 2020-09-15 | 2021-01-05 | 上海英基生物科技有限公司 | Method and kit for co-building DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) |
| WO2023240610A1 (en) * | 2022-06-17 | 2023-12-21 | 深圳华大智造科技股份有限公司 | Method for constructing single-stranded nucleic acid molecule sequencing library |
| CN116103383B (en) * | 2023-04-03 | 2023-06-20 | 北京百力格生物科技有限公司 | Method for identifying false base of NGS linker oligo and library thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463585A (en) * | 2014-09-12 | 2016-04-06 | 清华大学 | Method for constructing sequencing library based on single-stranded DNA molecule, and applications thereof |
| CN105506125A (en) * | 2016-01-12 | 2016-04-20 | 上海美吉生物医药科技有限公司 | DNA sequencing method and next generation sequencing library |
| CN108166068A (en) * | 2018-01-02 | 2018-06-15 | 上海美吉生物医药科技有限公司 | A kind of Novel DNA builds library kit and its application |
-
2019
- 2019-08-29 CN CN201910808734.5A patent/CN110607353B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463585A (en) * | 2014-09-12 | 2016-04-06 | 清华大学 | Method for constructing sequencing library based on single-stranded DNA molecule, and applications thereof |
| CN105506125A (en) * | 2016-01-12 | 2016-04-20 | 上海美吉生物医药科技有限公司 | DNA sequencing method and next generation sequencing library |
| CN108166068A (en) * | 2018-01-02 | 2018-06-15 | 上海美吉生物医药科技有限公司 | A kind of Novel DNA builds library kit and its application |
Non-Patent Citations (4)
| Title |
|---|
| Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections;Fredrik Salmén et al;《Nat Protoc》;20181130;13 * |
| Illumina sequencing library preparation for highly multiplexed target capture and sequencing;Matthias Meyer et al.;《Cold Spring Harb Protoc》;20100630;5 * |
| Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA;Marie-Theres Gansauge et al.;《Nat Protoc》;20130314;737-748 * |
| Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase;Gansauge MT et al.;《Nucleic Acids Research》;20170601;1-9 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110607353A (en) | 2019-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110607353B (en) | Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology | |
| EP3814494B1 (en) | High throughput assembly of nucleic acid molecules | |
| US20210189459A1 (en) | Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using crispr/cas system proteins | |
| CN105531375B (en) | Method for targeted genomic analysis | |
| EP3635114B1 (en) | Creation and use of guide nucleic acids | |
| US20120003657A1 (en) | Targeted sequencing library preparation by genomic dna circularization | |
| CN110699426B (en) | Gene target region enrichment method and kit | |
| US20150284712A1 (en) | Barcoding nucleic acids | |
| US10648031B2 (en) | Preparation of adapter-ligated amplicons | |
| CN102016068A (en) | Method of making a paired tag library for nucleic acid sequencing | |
| US12065684B2 (en) | Demand synthesis of polynucleotide sequences | |
| CN107475779A (en) | Library method for building up and its application suitable for unicellular RRBS sequencings | |
| WO2015050501A1 (en) | Amplification paralleled library enrichment | |
| CN110699425B (en) | Enrichment method and system of gene target region | |
| US20240271328A1 (en) | Rapid library construction for high throughput sequencing | |
| EP3198064B1 (en) | Methods for sample preparation | |
| EP3759243B1 (en) | Methods and compositions for enriching nucleic acids | |
| US20240271126A1 (en) | Oligo-modified nucleotide analogues for nucleic acid preparation | |
| CA2482425A1 (en) | Constant length signatures for parallel sequencing of polynucleotides | |
| CN111989406A (en) | Construction method of sequencing library | |
| WO2019090482A1 (en) | Second-generation high-throughput sequencing library construction method | |
| US20220282398A1 (en) | Universal blocking oligonucleotides for reduced off-target hybridization in hybridization capture methods | |
| WO2018009677A1 (en) | Fast target enrichment by multiplexed relay pcr with modified bubble primers | |
| AU2018279112B2 (en) | Creation and use of guide nucleic acids | |
| US20240368658A1 (en) | Demand Synthesis of Polynucleotide Sequences |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |