CN114686453A - Method and kit for constructing transcriptome sequencing library - Google Patents
Method and kit for constructing transcriptome sequencing library Download PDFInfo
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- CN114686453A CN114686453A CN202011586642.6A CN202011586642A CN114686453A CN 114686453 A CN114686453 A CN 114686453A CN 202011586642 A CN202011586642 A CN 202011586642A CN 114686453 A CN114686453 A CN 114686453A
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- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
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Abstract
The invention discloses a method and a kit for constructing a transcriptome sequencing library, relates to the technical field of sequencing, and particularly provides an enzyme composition which is used for one-step reaction of second strand cDNA synthesis, filling-up and end adding A.
Description
Technical Field
The invention belongs to the technical field of sequencing. More particularly, it relates to a method and a kit for constructing a transcriptome sequencing library.
Background
Transcriptome (transcriptome) broadly refers to the collection of all transcripts in a cell under certain physiological conditions, including messenger RNA, ribosomal RNA, transfer RNA, and non-coding RNA; in the narrow sense, refers to the collection of all mRNAs. Proteins are the main contributors to cell function, proteome is the most direct description of cell function and state, transcriptional composition is the main means to study gene expression, transcriptome is the necessary link of proteome linking genomic genetic information with biological function, and regulation of transcriptional level is the most studied at present and is also the most important regulation mode of organisms.
Transcriptome sequencing is the sum of all RNAs that a particular cell can transcribe in a functional state, mainly including mRNA and non-coding RNAs. Transcriptome research is the basis and starting point of gene function and structure research, and almost all transcript sequence information of a specific tissue or organ of a certain species under a certain state can be comprehensively and rapidly obtained through new-generation high-throughput sequencing, wherein the transcript sequence information comprises mRNA (messenger ribonucleic acid) of coded protein and various non-coded RNAs, expression abundance of different transcripts generated by gene alternative splicing and the like. While analyzing the structure and expression level of the transcript, unknown transcripts and rare transcripts are also discovered, so that important problems of life sciences such as gene expression difference, gene structure variation, molecular marker screening and the like are accurately analyzed, and the method is widely applied to the fields of basic research, clinical diagnosis, drug research and development and the like.
Transcripts of the sense and antisense strands are present in many gene regions, and most non-coding RNAs in particular are derived from the antisense strand. Strand-specific transcriptome sequencing refers to storing the orientation information of an RNA strand in a sequencing library during library construction, and data analysis after sequencing can determine whether the transcript is from a sense strand or an antisense strand. The advantages of strand-specific sequencing can lead to more accurate gene expression quantification, improved accuracy in finding circRNA, identification of non-coding antisense transcripts, better prediction of new transcripts, analysis of prokaryotic operons, and the like.
In the conventional construction process of the strand-specific transcriptome library, second strand cDNA synthesis, filling-in and terminal adding A are carried out in two steps, purification is needed in the middle, mRNA enrichment and interruption are needed, reverse transcription of first strand cDNA is needed, connection and joint are needed, purification, quality detection of the transcriptome library, sequencing on a computer and the like are needed; the library constructed by the process has the problems of complex reagent components, high cost, complex operation steps, long library construction time, low efficiency and the like.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide an enzyme composition.
Another object of the present invention is to provide the use of the enzyme composition in the construction of a transcriptome sequencing library and/or the preparation of a kit for the construction of a transcriptome sequencing library.
It is yet another object of the present invention to provide a method for constructing a transcriptome sequencing library.
It is still another object of the present invention to provide a kit for constructing a transcriptome sequencing library.
The invention is realized by the following technical scheme:
the invention provides an enzyme composition, which is characterized by consisting of a first DNA polymerase, RNaseH and a second DNA polymerase, wherein the second DNA polymerase is Taq DNA polymerase.
The application of the enzyme composition in construction of a transcriptome sequencing library and/or preparation of a kit for construction of a transcriptome sequencing library also falls within the scope of the present invention.
The invention also provides a method for constructing a transcriptome sequencing library, which comprises the following steps:
s1, fragmenting mRNA, and performing first strand cDNA synthesis by using first strand cDNA synthetase;
s2, performing second-strand cDNA synthesis, filling in and end-to-end reaction by using the enzyme composition;
and S3, connecting a joint, and purifying magnetic beads to obtain the transcriptome sequencing library.
In addition, the invention also provides a kit for constructing a transcriptome sequencing library, which comprises the enzyme composition.
The invention has the following beneficial effects:
the invention provides a method and a kit for constructing a transcriptome sequencing library. According to the invention, second-strand cDNA synthesis, filling in, and terminal addition A are combined into one step, the step only needs 3 enzymes (first DNA polymerase, RNase H and second DNA polymerase), and a purification step is not needed in the middle, so that the library building process can be efficiently carried out, the operation process is greatly simplified, the library building only needs 3.5 hours, the library building efficiency is improved, the consumption of reagents is reduced, the library building cost is reduced, the quality of the constructed transcriptome sequencing library is ensured, and the application prospect in constructing the transcriptome sequencing library and preparing the kit for constructing the transcriptome sequencing library is good.
Drawings
FIG. 1 is a schematic diagram of the construction of a transcriptome sequencing library according to examples 6-10 of the present invention; wherein "fragmentation and random primer" represents fragmentation and random primer; "First-Strand Synthesis" stands for First Strand cDNA Synthesis; "Second-Strand Synthesis & A labeling" stands for Second Strand cDNA Synthesis, end repair and addition of A; "adapter Ligation" represents a linker linkage; "PCR Amplification" stands for PCR Amplification.
FIG. 2 is a schematic diagram of comparative example 1 construction of a transcriptome sequencing library according to the present invention; wherein "fragmentation and random primer" represents fragmentation and random primer; "First-Strand Synthesis" stands for First Strand cDNA Synthesis; "Second-Strand Synthesis" stands for Second Strand cDNA Synthesis; "End Repair/A-tailing" stands for End Repair and adding A; "adapter Ligation" represents a linker linkage; "PCR Amplification" stands for PCR Amplification.
FIG. 3 is a diagram showing the results of quality control of the transcriptome sequencing library constructed in example 6 and comparative example 1; wherein "case 1" represents example 6; "case 2" represents comparative example 1.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise specified, reagents and materials used in the following embodiments are commercially available.
The invention relates to an enzyme composition, which consists of a first DNA polymerase, RNaseH and a second DNA polymerase, wherein the second DNA polymerase is Taq DNA polymerase.
In some embodiments, the enzyme activity concentration ratio of the first DNA polymerase, RNaseH and the second DNA polymerase is (0.02-0.3): (0.002-0.1): (0.01-0.2).
In an alternative embodiment, the enzyme activity concentration ratio of the first DNA polymerase, RNaseH and the second DNA polymerase is (0.05-0.2): (0.02-0.1): (0.04-0.2).
In an alternative embodiment, the enzyme activity concentration ratio of the first DNA polymerase, RNaseH, second DNA polymerase is 0.1:0.04: 0.1.
In some embodiments, the first DNA polymerase is a DNA polymerase having 5 '-3' polymerase activity.
In alternative embodiments, the first DNA polymerase further has 5 '-3' exo activity and/or 3 '-5' exo activity.
In some embodiments, the first DNA polymerase is any one or more of DNA polymerase I, DNA polymerase I (Klenow) large fragment, Klenow fragment (3'→ 5' exo-) or T4 DNA polymerase.
In alternative embodiments, the first DNA polymerase is DNA polymerase I.
In some embodiments, the enzyme composition is used for second strand cDNA synthesis, filling in, and end-plus-a one-step reactions.
The enzyme composition can be used for constructing a transcriptome sequencing library and/or preparing a kit for constructing the transcriptome sequencing library.
The invention also relates to a method for constructing a transcriptome sequencing library, which comprises the following steps:
s1, fragmenting mRNA, and performing first strand cDNA synthesis by using first strand synthetase;
s2, performing second-strand cDNA synthesis, filling in and end-to-end reaction by using the enzyme composition;
and S3, connecting the joints, and carrying out PCR amplification to obtain the transcriptome sequencing library.
In some embodiments, purification is performed after the linker is ligated in step S3; purification is performed after PCR amplification as described in step S3.
In an alternative embodiment, the purification is magnetic bead purification.
In some embodiments, the buffer used in the one-step reaction comprises 5-50mM buffer, 3-50mM Mg2+25-100mM monovalent cation, 5-20mM DTT, 0.1-0.5mM second dNTP;
the second dNTP is a mixture of dATP, dUTP/dTTP, dCTP and dGTP in a molar ratio of (2-10) to 1:1:1, and the pH value is 7.5-8.5.
For example, the buffer may have a concentration of 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, Mg2+The concentration of (B) may be 3mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, the concentration of monovalent cation may be 25mM, 35mM, 45mM, 55mM, 65mM, 75mM, 85mM, 95mM, 100mM, the concentration of DTT may be 5mM, 10mM, 15mM, 20mM, and the concentration of second dNTP may be 0.1mM, 0.14mM, 0.2mM, 0.3mM, 0.4mM, 0.5 mM.
In an alternative embodiment, the buffer used in the one-step reaction comprises 5-25mM buffer, 5-25mM Mg2+25-75mM monovalent cation, 5-15mM DTT, 0.15-0.4mM second dNTP;
the second dNTP is a mixture of dATP, dUTP/dTTP, dCTP and dGTP in a molar ratio of (5-10) to 1:1:1, and the pH value is 7.5-8.1.
In an alternative embodiment, the buffer used in the one-step reaction comprises 10mM buffer, 10mM Mg2+50mM monovalent cation, 10mM DTT, 0.2mM second dNTP;
the second dNTP was a mixture of dATP, dUTP/dTTP, dCTP and dGTP in a molar ratio of 7.5:1:1:1, pH 7.9.
In alternative embodiments, the buffer is Tris-HCl or PB.
In an alternative embodiment, the monovalent cation is Na+、K+Or Li+Any one or more of them.
In some casesIn embodiments, the fragmentation buffer of step S1 comprises 50-100mM Tris-HCl, 3-10mM MgSO40.1-15 mu M random primer, and pH 7.5-8.5.
In alternative embodiments, the fragmentation buffer of step S1 comprises 60-80mM Tris-HCl, 5-8mM MgSO4And 1-10 mu M of random primer, and the pH value is 8.2-8.5.
In some embodiments, the random primer of step S1 is a random hexamer primer.
In an alternative embodiment, the random hexamer primer of step S1 is a random hexamer primer with a 5' phosphorylation modification.
In some embodiments, the first strand cDNA synthetase of step S1 comprises RNasin and MMLV reverse transcriptase.
In an alternative embodiment, the concentrations of RNase and MMLV reverse transcriptase in the first strand cDNA synthesis system are 0.5-2U/. mu.l and 2.5-10U/. mu.l, respectively.
In an alternative embodiment, the concentrations of RNase and MMLV reverse transcriptase in the first strand cDNA synthesis system are 0.5-1U/. mu.l and 5-10U/. mu.l, respectively.
In some embodiments, the first strand cDNA synthesis buffer of step S1 comprises 50-100mM Tris-HCl, 5-30mM (NH)4)2SO425-100mM KCl, 5-50mM LiCl, 5-20mM DTT, 0.005% -0.2% Tween 20 and 0.1-0.5mM first dNTP;
the first dNTP is a mixture of dATP, dTTP, dCTP and dGTP (the molar ratio is 1:1:1:1), and the pH value is 7.5-8.5.
In alternative embodiments, the first strand cDNA synthesis buffer of step S1 comprises 60-80mM Tris-HCl, 5-15mM (NH)4)2SO450-80mM KCl, 10-30mM LiCl, 5-10mM DTT, 0.01-0.05% Tween 20 and 0.2-0.3mM first dNTP;
the first dNTP is a mixture of dATP, dTTP, dCTP and dGTP (the molar ratio is 1:1:1:1), and the pH value is 8.2-8.5.
In some embodiments, the ligase of step S3 is T4 DNA ligase.
In some embodiments, the concentration of the T4 DNA ligase in the ligation system is 6-30U/. mu.l.
In an alternative embodiment, the T4 DNA ligase is present in the ligation system at a concentration of 20-30U/. mu.l.
In some embodiments, the ligation buffer of step S3 comprises 50-100mM Tris-HCl, 5-20mM MgCl2、4％-20％PEG4000、0.1-1mM ATP，pH 7.5～8.5。
In alternative embodiments, the ligation buffer of step S3 comprises 50-80mM Tris-HCl, 5-10mM MgCl2、5％-10％PEG4000、0.2-0.5mM ATP，pH 7.5～8.0。
Methods for enriching mRNA that are conventional in the art can be used in the present invention.
In some embodiments, the method for enriching mRNA of step S1 is a poly (a) method or a ribosome RNA removal method.
Linkers conventional in the art may be used in the present invention.
In some embodiments, the joint of step S3 is an incomplete Y-shaped double-link joint or a U-shaped joint.
In the present invention, the PCR amplification in step S3 can be accomplished by a conventional method. U base digestion can be performed prior to PCR amplification; when the "U" bases are not digested with additional enzyme, the enzyme premix for PCR amplification can be selected to be Fapon 2 × Hammer-fidelity amplification PCR Mix or KaPa HiFi HotStart Ready Mix.
The respective modules of the Fapon Stranded Specific RNA Library Preparation Kit for Illumina (cat # MD079) are in the ranges of the components and the concentrations of the fragmentation buffer, first strand cDNA synthetase, first strand cDNA synthesis buffer, ligase, ligation buffer, etc. described above.
The invention also relates to a kit for constructing a transcriptome sequencing library, which comprises the enzyme composition.
Unless otherwise specified, the above "concentrations" refer to the final concentration of the system.
Some of the reagents used in the examples below were from the Fapon Stranded Specific RNA Library Preparation Kit for Illumina (cat. No. MD079) module, abbreviated as MD 079.
Example 1 enzyme composition
An enzyme composition comprises DNA polymerase I, RNaseH and Taq DNA polymeras with enzyme activity concentration ratio of 0.1:0.04: 0.1.
Example 2 enzyme composition
An enzyme composition comprises DNA polymerase I, RNaseH and Taq DNA polymeras with enzyme activity concentration ratio of 0.02:0.1: 0.01.
Example 3 enzyme composition
An enzyme composition comprises T4 DNA polymerase, RNaseH and Taq DNA polymerase with enzyme activity concentration ratio of 0.3:0.002: 0.02.
Example 4 enzyme composition
An enzyme composition comprises large fragment of DNA polymerase I (Klenow), RNaseH and Taq DNA polymerase with enzyme activity concentration ratio of 0.05:0.1: 0.04.
Example 5 enzyme composition
An enzyme composition comprises Klenow fragment (3'→ 5' exo-), RNaseH and Taq DNA polymerase, wherein the enzyme activity concentration ratio is 0.2:0.02: 0.2.
Example 6 construction of transcriptome sequencing libraries
The total RNA of the human culture cell sample is extracted by utilizing a commercial column extraction method extraction kit, and the complete condition of the RNA sample is detected by utilizing a Qsep 100 bioanalyzer. Selecting a sample with total RNA RIN >10, and using mRNA enriched by a commercial poly (A) method coupled oligo-dT magnetic bead enrichment extraction kit (poly (A) method) as an RNA sample. Then, mRNA was quantified using a Qubit2.0 fluorescence quantifier, 10ng of mRNA was reacted in each tube, and a transcriptome sequencing library was constructed. The method comprises the following specific steps:
1. mRNA fragmentation
10ng of mRNA sample was put into a 0.2ml PCR tube, 5. mu.l of fragmentation buffer (MD079) was added, DEPC was added to 10ul of water, mixed well, and heated at 95 ℃ for 5min in a PCR apparatus to perform mRNA fragmentation treatment.
2. First Strand cDNA Synthesis
Mu.l of first strand cDNA synthetase (MD079) and 1. mu.l of first strand cDNA synthesis buffer (MD079) were added to the fragmented mRNA, and DEPC was made up to 20ul in water, mixed well and run in a PCR machine set to the following conditions: keeping at 25 deg.C for 10min, 42 deg.C for 15min, 75 deg.C for 15min, and 4 deg.C.
3. Second strand cDNA, filling in, end adding A one-step reaction
Mu.l of the enzyme composition of example 1 (first DNA polymerase, RNaseH, second DNA polymerase) and 5. mu.l of buffer for one-step reaction were added to the first strand cDNA system, DEPC water was made up to 50ul, mixed well and run in a PCR apparatus set to the following conditions: 30min at 16 ℃, 30min at 65 ℃ and 4 ℃.
The enzyme activity concentrations of the first DNA polymerase, the RNaseH and the second DNA polymerase in the one-step reaction system are 0.1U/mul, 0.04U/mul and 0.1U/mul in sequence.
The buffer used in the one-step reaction comprises 10mM Tris-HCl and 10mM MgCl250mM NaCl, 10mM DTT, 0.2mM second dNTP;
the second dNTP was a mixture of dATP, dUTP, dCTP and dGTP in a molar ratio of 7.5:1:1:1, pH 7.9.
4. Joint connection
Mu.l ligase (MD079) and 34. mu.l ligation buffer (MD079) were added to the reaction product of the first step,
2.5. mu.l of Singleplex oligonucleotides for Illumina (NEB # E7350S) 25-fold dilution, made up to 100. mu.l without nuclease water, mixed well and run in a PCR apparatus set to the following conditions: 15min at 20 ℃.
5. Ligation product magnetic bead purification
Transferring the ligation product to a 1.5ml centrifuge tube, adding 90. mu.l of AMPure magnetic beads (0.9X), mixing, incubating at room temperature for 5min, placing on a magnetic rack, and removing the supernatant after the solution is clarified. The beads were rinsed by adding 200. mu.L of 80% ethanol and after the solution was clarified, the supernatant was removed. The 80% ethanol rinse was repeated once. Keeping the centrifugal tube on the magnetic frame all the time, and opening the cover at room temperature to dry the magnetic beads for about 2-5 min. Adding 21.5 mu L of nuclease-free water into the centrifuge tube, fully mixing, standing at room temperature for 2min, placing on a magnetic frame, and carefully sucking 20 mu L of purified product into a new PCR tube after the solution is clarified.
6. PCR amplification
To the purified ligation product, 25. mu.L of Fapon 2 × Hammer-fidelity PCR Mix (MD021), 2.5. mu.L of NEB Next Universal PCR Primer (NEB # E7350S), 2.5. mu.L of NEB Next Index Primer (NEB # E7350S) were added, and after vortexing the system, the system was centrifuged and run in a PCR machine set to the following conditions: 45s at 98 ℃ (15 s at 98 ℃, 30s at 65 ℃, 30s at 72 ℃) in 10cycles, 5min at 72 ℃, and keeping at 4 ℃.
7. Magnetic bead purification of PCR product to obtain transcriptome sequencing library
The PCR product was transferred to a 1.5ml centrifuge tube, 45 μ l AMPure magnetic beads (0.9 ×) were added and gently pipetted and mixed using a pipette, then incubated at room temperature for 5min and placed on a magnetic rack, and after the solution cleared, the supernatant was removed. The beads were rinsed by adding 200. mu.L of freshly prepared 80% ethanol and after the solution cleared, the supernatant was removed. The 80% ethanol rinse was repeated once. Keeping the centrifugal tube on the magnetic frame all the time, and opening the cover at room temperature to dry the magnetic beads for about 2-5 min. Adding 21.5 mu L of nuclease-free water into a centrifuge tube, gently sucking and beating by using a pipette, fully and uniformly mixing, standing at room temperature for 2min, placing on a magnetic frame, and obtaining a supernatant which is a transcriptome sequencing library product after the solution is clarified.
Example 7 construction of transcriptome sequencing libraries
This example 7 differs from example 6 in the following way:
mRNA enriched by the ribosome RNA removal method in the step 1;
the enzymes used in the one-step reaction in the step 3 are the enzyme compositions (the first DNA polymerase, RNaseH and the second DNA polymerase) of the embodiment 2, the enzyme activity concentrations of the first DNA polymerase, the RNaseH and the second DNA polymerase in the one-step reaction system are 0.02U/microliter, 0.1U/microliter and 0.01U/microliter in sequence, and the buffer used in the one-step reaction comprises 5mM PB and 25mM MgCl225mM KCl, 15mM DTT, 0.15mM second dNTP;
the second dNTP is a mixture of dATP, dTTP, dCTP and dGTP in a molar ratio of 10:1:1:1, pH 7.5;
the joint used in the step 4 is Hieff
96 Single Index Primer Kit for
Set 1(48 species) (12611ES 02).
The Fapon 2 × Hammer-fidelity amplification PCR Mix (MD021) in step 6 is replaced by KAPA HiFi HotStart Ready Mix (KK2600), and the NEB Next Universal PCR Primer and NEB Next Index Primer are replaced by Hieff
Universal Primer (12611ES02) and Hieff
Index Primer(12611ES02)。
Example 8 construction of transcriptome sequencing libraries
This example 8 differs from example 6 in the following way:
the enzymes used in the one-step reaction in the step 3 are the enzyme compositions (the first DNA polymerase, the RNaseH and the second DNA polymerase) in the example 3, the enzyme activity concentrations of the first DNA polymerase, the RNaseH and the second DNA polymerase in the one-step reaction system are 0.3U/microliter, 0.002U/microliter and 0.02U/microliter in sequence, and the buffer used in the one-step reaction comprises 25mM Tris-HCl and 5mM MgCl275mM LiCl, 5mM DTT, 0.4mM second dNTP;
the second dNTP was a mixture of dATP, dUTP, dCTP and dGTP in a molar ratio of 5:1:1:1, pH 8.1.
Example 9 construction of transcriptome sequencing libraries
This example 9 differs from example 6 in the following way:
the enzyme used in the one-step reaction in the step 3 is the enzyme composition (the first DNA polymerase, RNaseH, the second DNA polymerase) of the embodiment 4, the enzyme activity concentrations of the first DNA polymerase, the RNaseH, the second DNA polymerase in the one-step reaction system are 0.05U/μ l, 0.1U/μ l, 0.04U/μ l in sequence, and the buffer used in the one-step reaction comprises 5mM Tris-HCl and 50mM MgCl225mM NaCl, 20mM DTT, 0.1mM second dNTP;
the second dNTP was a mixture of dATP, dTTP, dCTP and dGTP in a molar ratio of 10:1:1:1, pH 7.5.
Example 10 construction of transcriptome sequencing libraries
This embodiment 10 differs from embodiment 6 in the following way:
the enzymes used in the one-step reaction in the step 3 are the enzyme compositions (the first DNA polymerase, the RNaseH and the second DNA polymerase) in the embodiment 5, the enzyme activity concentrations of the first DNA polymerase, the RNaseH and the second DNA polymerase in the one-step reaction system are 0.2U/microliter, 0.02U/microliter and 0.2U/microliter in sequence, and the buffer used in the one-step reaction comprises 50mM PB and 3mM MgCl2100mM KCl, 5mM DTT, 0.5mM second dNTP;
the second dNTP was a mixture of dATP, dUTP, dCTP and dGTP in a molar ratio of 2:1:1:1, pH 8.5.
The schematic diagrams of the construction of the transcriptome sequencing libraries of examples 6-10 above are shown in FIG. 1.
Comparative example 1
Comparative example 1 a schematic diagram of the construction of a transcriptome sequencing library is shown in figure 2.
Comparative example 1 differs from example 6 only in step 3:
and 3, the second strand cDNA synthesis, the filling-in and the end A addition in the step 3 need to be completed step by step (6 enzymes are needed and the used buffer is complex), and the middle needs to be purified, namely, the second strand cDNA synthesis is firstly carried out, then the magnetic bead purification and recovery are carried out, and then the filling-in and the end A addition are completed. The method comprises the following specific steps:
second strand cDNA synthesis requires 2 enzymes: 0.0625U/. mu.l DNA polymerase I and 0.025U/. mu.l RNaseH.
The second strand cDNA synthesis buffer comprises 10mM Tris-HCl, 10mM MgCl250mM NaCl, 10mM DTT, 0.2mM second dNTP, wherein the second dNTP is a mixture of dATP, dUTP, dCTP and dGTP in a molar ratio of 1:1:1:1, pH 7.9;
after the second strand cDNA synthesis product was recovered by AMPure magnetic bead (1.8 ×), the completion, end-plus-A synthesis required 4 enzymes: 2U/. mu. l T4 PNK, 0.15U/. mu. l T4 DNA polymerase, 0.1U/. mu.l Klenow fragment (3'→ 5' exo +), 0.06U/. mu.l Taq DNA polymerase;
the filling and end-to-end A buffer comprises 60mM Tris-HCl, 20mM MgSO4、10mM NaCl、1mM ATP、0.2mM of a second dNTP, wherein the second dNTP is a mixture of dATP, dTTP, dCTP and dGTP at a ratio of 7.5:1:1:1, pH 7.3.
Comparative example 2
Comparative example 2 differs from example 6 only in step 3:
the enzyme composition used in the one-step reaction of step 3 consists of 6 enzymes, and the 6 enzymes are respectively: 0.1U/. mu.l DNA polymerase I, 0.04U/. mu.l RNaseH, 2U/. mu. l T4 PNK, 0.15U/. mu. l T4 DNA polymerase, 0.1U/. mu.l Klenow fragment (3'→ 5' exo +), 0.06U/. mu.l Taq DNA polymerase.
Comparative example 3
Comparative example 3 differs from example 6 only in step 3:
the enzyme composition used in the one-step reaction of step 3 consists of 5 enzymes, 5 enzymes are respectively: 0.1U/. mu.l DNA polymerase I, 0.04U/. mu.l RNaseH, 2U/. mu. l T4 PNK, 0.1U/. mu.l Klenow fragment (3'→ 5' exo +), 0.06U/. mu.l Taq DNA polymerase.
Comparative example 4
Comparative example 4 differs from example 6 only in step 3:
the enzyme composition used in the one-step reaction of step 3 consists of 4 enzymes, wherein the 4 enzymes are respectively: 0.1U/. mu.l DNA polymerase I, 0.04U/. mu.l RNaseH, 0.1U/. mu.l Klenow fragment (3'→ 5' exo +), 0.06U/. mu.l Taq DNA polymerase.
Performing quality inspection on the library: the transcriptome sequencing libraries constructed in examples 6-10 and comparative examples 1-4 above were subjected to concentration determination using a qubit2.0 fluorescence quantifier, and the size was detected using a Qsep 100 bioanalyzer.
The quality inspection results of the transcriptome sequencing library constructed in the embodiments 6 to 10 and the comparative examples 1 to 4 are shown in table 1 and fig. 3, the computer-on results of the library are shown in table 2, and the results show that the library-constructing time of the invention is 3.5h, the operation process is simplified, and the library-constructing yield is remarkably improved, wherein the library-constructing time is high-quality chain-specific transcriptome sequencing library successfully prepared in the embodiments of the invention; compared with comparative examples 2-4, the invention ensures the yield of second strand cDNA by reasonably screening the combined enzyme system, thereby ensuring the output of high-quality RNA library.
Theoretically, when the second strand cDNA synthesis, magnetic bead purification and recovery, filling in, and end adding A in the conventional transcriptome library construction process steps are combined into a one-step reaction, 6 enzymes, second strand cDNA synthesis buffer and filling in, end adding A buffer are needed to work together; the present inventors have surprisingly found that simply stacking 6 enzymes (comparative example 2), selecting 5 enzymes (comparative example 3) or 4 enzymes (comparative example 4) does not yield a high quality RNA library; when 3 enzymes were selected and the amounts of enzymes used were adjusted appropriately (examples 6-10), high quality RNA libraries were obtained with the highest library yields.
TABLE 1 results of quality control of transcriptome sequencing libraries constructed in examples 6 to 10 and comparative examples 1 to 4
TABLE 2 results on the library
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. An enzyme composition, wherein the enzyme composition comprises a first DNA polymerase, RNaseH, and a second DNA polymerase, and the second DNA polymerase is Taq DNA polymerase.
2. The enzyme composition of claim 1, wherein the enzyme activity concentration ratio of the first DNA polymerase, RNaseH and the second DNA polymerase is (0.02-0.3): (0.002-0.1): (0.01-0.2).
3. The enzyme composition of claim 1, wherein the first DNA polymerase is a DNA polymerase having 5 '-3' polymerase activity;
optionally, the first DNA polymerase further has 5 '-3' exo activity and/or 3 '-5' exo activity.
4. The enzyme composition according to claim 1, wherein the first DNA polymerase is any one or more of DNA polymerase I, DNA polymerase I (Klenow) large fragment, Klenow fragment (3'→ 5' exo-) or T4 DNA polymerase.
5. The enzyme composition according to any one of claims 1 to 4, wherein the enzyme composition is used for second strand cDNA synthesis, filling-in, and end-plus-A one-step reaction.
6. Use of the enzyme composition according to any one of claims 1 to 5 for constructing a transcriptome sequencing library and/or for preparing a kit for constructing a transcriptome sequencing library.
7. A method of constructing a transcriptome sequencing library comprising the steps of:
s1, fragmenting mRNA, and performing first strand cDNA synthesis by using first strand cDNA synthetase;
s2, performing second strand cDNA synthesis, filling in, and adding A at the tail end by using the enzyme composition of any one of claims 1 to 5;
and S3, connecting the joints, and carrying out PCR amplification to obtain the transcriptome sequencing library.
8. The method of claim 7, wherein the linker is ligated and then purified in step S3 and/or the PCR amplification is followed by purification in step S3.
9. The method of claim 7, wherein the buffer used in the one-step reaction of step S2 comprises 10-50mM buffer and 10-50mM Mg2+25-100mM monovalent cation, 5-20mM DTT, 0.1-0.5mM second dNTP, pH 7.5-8.5;
the second dNTP is a mixture of dATP, dUTP/dTTP, dCTP and dGTP in a molar ratio of (2-10):1:1: 1.
10. A kit for constructing a transcriptome sequencing library, comprising the enzyme composition of any one of claims 1 to 5.
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