Template conversion method in cDNA library building

Abstract

The invention discloses a template conversion method in cDNA (complementary deoxyribonucleic acid) library establishment. The method comprises the following steps: S1, mixing an RNA template and a reverse transcription primer, and annealing to obtain an RNA template / reverse transcription primer hybrid chain; s2, carrying out reverse transcription on the RNA template / reverse transcription primer hybrid chain by adopting II-type intron coding reverse transcriptase to obtain a reverse transcription product; and S3, mixing the reverse transcription product, a TSO primer and M-MLV reverse transcriptase, and carrying out template conversion. According to the method, the template conversion efficiency of the cDNA with the length of more than 1kb is remarkably improved, and the template conversion efficiency of the cDNA with the length of less than 1kb is not influenced; meanwhile, the reaction time of reverse transcription and template exchange in the cDNA library construction process is also greatly shortened.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a template conversion method in cDNA library construction. Background Art

[0002] Full-length transcriptome / single-cell full-length transcriptome sequencing is one of the key tools for RNA research. Since RNA is relatively easy to degrade, it is often reverse transcribed into cDNA (Complementary DNA) in RNA research. However, the traditional reverse transcription method often results in the loss of transcription information at the 5' end of cDNA. In 2001, SMART technology made it possible to produce a complete cDNA library even for single-cell RNA samples by utilizing the characteristics of M-MLV reverse transcriptase and combining the ingenious design of template switch oligonucleotides (TSO). First, when M-MLV reverse transcriptase relies on its reverse transcriptase activity to reach the 5' end of the RNA template, its terminal transferase activity will add additional deoxycytidine (dC) to the 3' end of the newly synthesized cDNA chain. The 3' end of TSO has three riboguanosines (rGrGrG), and these consecutive rG bases will form base complementary pairing with the dC attached to the 3' end of the cDNA molecule, thereby inducing the reverse transcriptase from RNA to TSO and continuing to synthesize cDNA to the 5' end of TSO. The resulting cDNA not only contains the complete 5' end of the transcript, but also can amplify the entire transcript independently of the cDNA sequence, significantly enhancing the integrity of the data.

[0003] We are still trying to improve the TSO design of template exchange and screen reverse transcriptases with high processivity and high terminal transferase activity to further improve the quality and construction efficiency of cDNA libraries. Summary of the invention

[0004] In view of the above problems, the present invention provides a template switching method in cDNA library construction, comprising:

[0005] S1. Mixing RNA template and reverse transcription primer to anneal to obtain RNA template / reverse transcription primer hybrid chain;

[0006] S2. using a group II intron-encoded reverse transcriptase to reverse transcribe the RNA template / reverse transcription primer hybrid chain to obtain a reverse transcription product;

[0007] S3. Mix the reverse transcription product, TSO primer and M-MLV reverse transcriptase for template switching.

[0008] Optionally, according to the above template switching method, the group II intron encoding reverse transcriptase is selected from Reverse Transcriptase and / or Marothon RT.

[0009] Optionally, according to the above template switching method, the M-MLV reverse transcriptase is selected from at least one of Maxima H Minus, SuperScript IV and SuperScriptII.

[0010] Optionally, according to the above template switching method, the TSO primer sequence is shown as SEQ ID NO.2.

[0011] Optionally, according to the above template switching method, the reverse transcription primer sequence is shown as SEQ ID NO.1.

[0012] The present invention also provides a cDNA library construction method, comprising:

[0013] (1) implementing the above-mentioned template conversion method;

[0014] (2) Using the product obtained in (1), prepare a nanopore sequencing library.

[0015] Optionally, according to the above-mentioned cDNA library construction method, the step (2) comprises

[0016] (21) performing PCR amplification on the product obtained in (1) to obtain a PCR product;

[0017] (22) performing an un-trimmed modification on the PCR product to obtain an un-trimmed product;

[0018] (23) connecting the unrepaired product to the adapter complex to obtain a connection product;

[0019] Preferably, the method further comprises (24) purifying the ligation product.

[0020] Optionally, according to the above-mentioned cDNA library construction method, the final modification in step (22) is to add A to the 3' end of the PCR product and phosphorylate the 5' end.

[0021] Optionally, according to the above-mentioned cDNA library construction method, the adapter complex includes a non-RNA polynucleotide sequencing sequence and a helicase. The non-RNA polynucleotide includes a first segment for guiding the fragment to be sequenced into the nanopore, a second segment for binding the helicase, a third segment for controlling the moving speed of the helicase along the fragment to be sequenced, and a fourth segment for connecting the sequencing fragment.

[0022] Optionally, according to the above-mentioned cDNA library construction method, the primers used in the PCR in step (21) are a fragment of the reverse transcription primer with a length of 20-30 nt, and a fragment of the TSO primer with a length of 20-30 nt.

[0023] Group II introns encode reverse transcriptase (e.g. Reverse Transcriptase, MarothonRT) shows stronger continuous synthesis ability, thermal stability, and inhibitor tolerance during RNA reverse transcription to cDNA synthesis, and is particularly suitable for reverse transcription and cDNA synthesis of long transcripts, complex secondary structures, and RNA samples containing inhibitory components, which improves the 5'end sequencing coverage of long transcripts. Therefore, it has a great advantage in synthesizing a complete single-strand cDNA. The terminal transferase activity of M-MLV reverse transcriptase (such as Maxima H Minus, SuperScriptIV, SuperScriptII) ensures the template conversion process.

[0024] The present invention combines the high processivity of the reverse transcriptase encoded by the type II intron and the terminal transferase activity of the M-MLV reverse transcriptase, significantly improving the template conversion efficiency of cDNAs with a length of more than 1 kb, and does not affect the template conversion efficiency of cDNAs with a length of less than 1 kb; it also greatly shortens the reaction time of reverse transcription and template exchange during the construction of the cDNA library. The embodiments of the present invention, in particular, compare the full-length transcriptome sequencing of the nanopore. The combination of ReverseTranscriptase and Maxima H Minus can improve the efficiency of template conversion. This discovery optimizes the library construction method for full-length transcriptome sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the agarose electrophoresis detection principle.

[0026] Figure 2 This is the test result of Example 1.

[0027] Figure 3 Figure 1 is a flowchart of nanopore sequencing.

[0028] Figure 4 This is the structure of a nanopore sequencer.

[0029] Figure 5A Example 2 The length distribution of cDNA synthesized by Reverse Transcriptase and Maxima H Minus during reverse transcription and template switching, obtained by nanopore sequencing.

[0030] Figure 5B This is a length distribution diagram obtained by nanopore sequencing of cDNA synthesized by Maxima H Minus and Maxima H Minus participating in the reverse transcription and template switching process in Example 2.

[0031] Figure 5C This is a comparison chart of the template conversion efficiency of RNA of various lengths in the range of 0.5-9k in the two groups of tests in Example 2. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0034] Example 1 Detection of template exchange efficiency by agarose gel electrophoresis

[0035] The detection principle of this embodiment is as follows Figure 1As shown, a poly(A) RNA with 20 bases at the 3' end was used as a template, and the reverse transcription process used Oligo(dT) Primer: 5'-gttttcgcatttatcgtgaaacgctttcgTTTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO.1) as a reverse transcription primer. The Oligo(dT) primer was annealed and bound to the poly(A) tail of the RNA. TSO Primer: 5'-dSp / CTACACGACGCTCTTCCGATCTATrGrG+G / NH2-C6-3' (SEQ ID NO.2) is a template switching primer, using LNA locked nucleic acid (rGrG+G, rG refers to riboguanosine, +G refers to locked nucleic acid modification) to increase the thermal stability of nucleic acid duplexes and improve the specificity of hybridization, promoting template switching; dSp (dspacer (Abasic site) THF (Tetrahydrofuran) tetrahydrofuran modification) is used to introduce an empty deoxynucleotide without a base at the 5' end of TSO, and the reverse transcriptase will terminate the reverse transcription reaction when encountering this site; NH2-C6 (3'Amino modifier C6) 3' end amino modification, C6 represents the number of carbon atoms of the methylene straight chain between NH2 and DNA, and the carbon chain plays a role in increasing the spatial distance. This modification is used to block the 3' end of TSO. The template is reverse transcribed and template switched to obtain RNA / DNA hybrid chains. Finally, the template conversion efficiency was quantified by PCR amplification. The amount of amplified product from primer pair ① and primer ③ of group A represented the amount of reverse transcription product, and the amount of amplified product from primer pair ② and primer ③ of group B represented the amount of template conversion product. The conversion efficiency was the percentage of the brightness of the electrophoretic bands of the amplified products from primer pair B and primer pair A.

[0036] This example uses three groups of reverse transcriptases to participate in the reverse transcription and template switching process, namely ReverseTranscriptase (Induro, NEB) and Maxima H Minus, Reverse Transcriptase and IV Reverse Transcriptase (SSⅣ, Thermo), Maxima H Minus Reverse Transcriptase (Maxima H Minus, Thermo) and Maxima H Minus. The template conversion efficiency of the three combinations was detected by agarose gel electrophoresis. The specific operation is as follows:

[0037] 1) First-strand cDNA synthesis and template switching process

[0038] a) Induro and Maxima H Minus combination

[0039] Take 500 ng RNA Marker (Applied Biosystems (AM7150), i.e. poly(A) RNA in the table below) and add each component to an enzyme-free PCR tube according to the table below:

[0040] Table 1

[0041] Components Dosage (μL) Oligo(dT)Primer(100μM) 1μL 10mM dNTP mix (10mM each) 1μL poly(A)RNA to 500ng DEPC-treated H2O to 10μL

[0042] Mix well, put into PCR instrument, run program: 65℃, 5min; then incubate the PCR tube on ice for 3min. Continue to add the remaining components according to the table below:

[0043] Table 2

[0044] Components Dosage (μL) 5X Induro RT Reaction Buffer 4μL RNase Inhibitor,Murine(40U / μL) 0.2μL Induro Reverse Transcriptase(200U / μL) 1μL DEPC-treated H2O 4.8μL

[0045] Mix well, place it in a PCR instrument, and run the program: 55°C, 15min.

[0046] After the above reaction is completed, the Induro reverse transcription product is obtained. Take 10 μl and add each component according to the following table into the enzyme-free PCR tube:

[0047] Table 3

[0048]

[0049]

[0050] Mix well, put into PCR instrument, run the following program to complete template conversion.

[0051]

[0052] b) Induro and SSⅣ combination

[0053] Take 500 ng RNA Marker (i.e. poly(A) RNA in the table below) and add each component to an enzyme-free PCR tube according to the table below:

[0054] Table 4

[0055] Components Dosage (μL) Oligo(dT)Primer(100μM) 1μL 10mM dNTP mix (10mM each) 1μL poly(A)RNA to 500ng DEPC-treated H2O to 10μL

[0056] Mix well, put into PCR instrument, run program: 65℃, 5min; then incubate the PCR tube on ice for 3min. Continue to add the remaining components according to the table below:

[0057] Table 5

[0058] Components Dosage (μL) 5X Induro RT Reaction Buffer 4μL RNase Inhibitor,Murine(40U / μL) 0.2μL Induro Reverse Transcriptase(200U / μL) 1μL DEPC-treated H2O 4.8μL

[0059] Mix well, place it in a PCR instrument, and run the program: 55°C, 15min.

[0060] After the above reaction is completed, the Induro reverse transcription product is obtained. Take 10 μl and add each component according to the following table into the enzyme-free PCR tube:

[0061] Table 6

[0062] Components Dosage (μL) Induro reverse transcription product 10μL 5M Betaine 4μL TSO Primer+MgCl2 1μL <![CDATA[5X SuperScript TM IV Reaction Buffer]]> 2μL <![CDATA[SuperScript TM IV reverse transcriptase (200U / μL)]]> 1μL 100mM DTT 0.5μL DEPC-treated H2O 1.5μL

[0063] Mix well, put into PCR instrument, run the following program to complete template conversion.

[0064]

[0065] c) Maxima H Minus and Maxima H Minus

[0066] Take 500 ng RNA Marker (i.e. poly(A) RNA in the table below) and add each component to an enzyme-free PCR tube according to the table below:

[0067] Table 7

[0068] Components Dosage (μL) Oligo(dT)Primer(100μM) 0.5μL 10mM dNTP mix (10mM each) 1μL poly(A)RNA to 500ng DEPC-treated H2O to 8.5μL

[0069] Mix well, put into PCR instrument, run program: 65℃5min; then incubate the PCR tube on ice for 3min. Continue to add the remaining components according to the table below:

[0070] Table 8

[0071]

[0072]

[0073] Mix well, then place it in a PCR instrument and run the following program to complete single-strand cDNA synthesis and template switching.

[0074]

[0075] 2) Agarose gel electrophoresis was used to detect the template exchange efficiency of the three combinations

[0076] The RNA strand in the RNA / DNA hybrid strand was digested using RNase H (NEB M0297s); the digested product was purified using 1.4× magnetic beads (Beckman A63882 AMPure XP); and the template conversion efficiency was quantified by PCR amplification. The magnetic bead purified product of this embodiment was amplified using the primer pair A (5'-CTACACGACGCTCTTCCGATCT-3' (SEQ ID NO.3) with 5'-gttttcgcatttatcgtgaaacgctttcg-3' (SEQ ID NO.4)) and the primer pair B (5'-ggtgatgcctattaatttagttccatc-3' (SEQ ID NO.5)) with 5'-gttttcgcatttatcgtgaaacgctttcg-3').

[0077] Figure 2 Figure 1 is the agarose gel electrophoresis result of the amplified product. A is the amplified product band obtained by PCR using primer pair A, and B is the amplified product band obtained by PCR using primer pair B. ImageJ was used to quantify the ligation efficiency. The combination of Reverse Transcriptase and Maxima H Minus (I+M in the figure) is the best among the three combinations, with a template conversion efficiency of 93%. The second best combination is the combination of Maxima H Minus and Maxima H Minus (M+M in the figure), with a template conversion efficiency of 78%. Reverse Transcriptase and The template conversion efficiency of IV ReverseTranscriptase combination (I+IV in the figure) is 71%.

[0078] Example 2 Comparison of template switching efficiency of different methods by nanopore full-length transcriptome sequencing using RNA marker as the starting sample

[0079] The nanopore sequencing method used in this example is as follows Figure 3 As shown. The cDNA product obtained after reverse transcription and template switching of the RNA template (RNA with poly(A) tail) is used to prepare a library, and the library is sequenced by nanopore sequencing.

[0080] RNA Millennium TMThe markers are composed of a mixture of 10 different lengths of RNA, with sizes of 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 9k; and each length of RNA has a 20-base poly(A) tail at the 3' end. This example uses it as the starting sample and uses the nanopore full-length transcriptome sequencing method to compare The template conversion efficiency of the two groups of Reverse Transcriptase and Maxima H Minus, and the two groups of Maxima H Minus and Maxima H Minus.

[0081] 1) First-strand cDNA synthesis and template switching

[0082] See method a) in Example 1 to obtain a cDNA product of Induro and Maxima H Minus; and method c) in Example 1 to obtain a cDNA product of Maxima H Minus and Maxima H Minus.

[0083] 2) The cDNA products obtained in step 1) of this example were PCR amplified and purified.

[0084] Specific operation: Add each component in sequence according to the table below;

[0085] Table 9

[0086] Components Dosage (μL) cDNA products 100ng KAPA HiFi HotStart ReadyMix 25μL Primer 1 (10 μM) 1μL Primer 2 (10 μM) 1μL DEPC-treated H2O to 50μL

[0087] Primers were matched with 5'-CTACACGACGCTCTTCCGATCT-3' (Primer 1) and 5'-gttttcgcatttatcgtgaaacgctttcg-3' (Primer 2), annealing temperature was 60°C, extension time was 6 min, 12 cycles were run. After amplification, purification was performed using 1x DNA purification magnetic beads.

[0088] 3) The PCR product obtained in step 2) of this example was terminally trimmed using a conventional library construction kit (Cat. No. PY-DNA101, Beijing Puyi Biotechnology Co., Ltd.) to add an A at the 3' end and phosphorylate the 5' end for subsequent ligation with the adapter complex.

[0089] Specific operation: Add each component in sequence according to the table below, and run the program: 20℃, 5min; 65℃, 5min;

[0090] Table 10

[0091]

[0092]

[0093] After the reaction is complete, purify the DNA using 1x DNA purification magnetic beads.

[0094] 4) Connecting the unrepaired product obtained in step 3) of this example to the linker complex and purifying it.

[0095] The conventional library construction kit (Cat. No. PY-DNA101, Beijing Puyi Biotechnology Co., Ltd.) was used to connect and purify the sample adapter complex to complete the construction of the library. The detailed operation is as follows

[0096] 1. Prepare the following reaction system in a 1.5 mL centrifuge tube:

[0097] Table 11

[0098] Components volume Unfinished product 1000ng Ligation Buffer 25μL Adaptor complex 5μL DNA ligase 8μL <![CDATA[Nuclease-free H2O]]> up to100

[0099] After gently pipetting to mix, do not shake, place the centrifuge tube in a 20℃ metal bath and incubate for 15 minutes.

[0100] 2. Add 1 volume of resuspended magnetic beads to the centrifuge tube and mix well by pipetting, then incubate at room temperature for 10 minutes.

[0101] 3. Place the centrifuge tube on the magnetic rack to separate the magnetic beads for about 5-8 minutes. After the solution is clear, carefully remove the supernatant and discard it;

[0102] 4. Remove the centrifuge tube and add 200 μL fragment recovery buffer (AWB) to resuspend the magnetic beads, then place the centrifuge tube on the magnetic stand to separate the magnetic beads for about 3-5 minutes. After the solution is clear, carefully remove the supernatant and discard it. Repeat this step once. When removing the supernatant for the second time, try to remove all the residual liquid;

[0103] 5. Remove the centrifuge tube from the magnetic stand, add 15-30 μL elution buffer (EB) to resuspend the magnetic beads, and incubate at 37°C for 10 min;

[0104] 6. Separate the magnetic beads on the magnetic rack for about 2 minutes. After the solution is clear, carefully transfer the supernatant to a new centrifuge tube, taking care not to suck up the magnetic beads.

[0105] 7. Use Qubit fluorescence quantification instrument to detect library concentration and take 200ng library sample for sequencing.

[0106] 5) Sequencing on a machine (the structure of the nanopore sequencer used is as follows Figure 4 (shown)

[0107] The library samples were sequenced using the nanopore gene sequencing platform PolyseqOne (Cat. No. PY-NSRO01, Beijing Puyi Biotechnology Co., Ltd.).

[0108] The results of this example are as follows Figures 5A-5C shown. Figure 5A for The length distribution of cDNA synthesized by Reverse Transcriptase and MaximaH Minus during reverse transcription and template switching, obtained by nanopore sequencing. Figure 5B The length distribution of cDNA synthesized by Maxima H Minus and Maxima H Minus participating in the reverse transcription and template switching process was obtained by nanopore sequencing. Figure 5C The above two groups of tests compare the template conversion efficiency of RNA of different lengths in the range of 0.5-9k. The results show that The proportion of cDNAs of various lengths in the range of 0.5-9k in the combination of Reverse Transcriptase and Maxima H Minus was higher than that of Maxima H Minus and Maxima H Minus. It also verified that the combination of Induro and Maxima H Minus improved the efficiency of template conversion. This finding not only shortened the library construction time, but also helped to increase the read length of full-length transcriptome sequencing.

[0109] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A template switching method in cDNA library construction, characterized in that: include S1. Mixing RNA template and reverse transcription primer to anneal to obtain RNA template / reverse transcription primer hybrid chain; S2. using a group II intron-encoded reverse transcriptase to reverse transcribe the RNA template / reverse transcription primer hybrid chain to obtain a reverse transcription product; S3. Mix the reverse transcription product, TSO primer and M-MLV reverse transcriptase for template switching.

2. The template conversion method according to claim 1, characterized in that: The group II intron encodes a reverse transcriptase selected from At least one of Reverse Transcriptase and / or Marothon RT.

3. The template conversion method according to claim 1, characterized in that: The M-MLV reverse transcriptase is selected from at least one of MaximaH Minus, SuperScript IV and SuperScriptII.

4. The template conversion method according to claim 1, characterized in that: The TSO primer sequence is shown in SEQ ID NO.

2.

5. The template conversion method according to claim 1, characterized in that: The reverse transcription primer sequence is shown in SEQ ID NO.

1.

6. A cDNA library construction method comprising: (1) Implementing the template conversion method described in claim 1; (2) Using the product obtained in (1), prepare a nanopore sequencing library.

7. The cDNA library construction method according to claim 6, characterized in that: The step (2) comprises (21) performing PCR amplification on the product obtained in (1) to obtain a PCR product; (22) performing an un-trimmed modification on the PCR product to obtain an un-trimmed product; (23) connecting the unrepaired product to the adapter complex to obtain a connection product; Preferably, the method further comprises (24) purifying the ligation product.

8. The cDNA library construction method according to claim 7, characterized in that: The final modification in step (22) is to add A to the 3' end of the PCR product and phosphorylate the 5' end.

9. The cDNA library construction method according to claim 7, characterized in that: The adaptor complex includes an adaptor sequence and a helicase.

10. The cDNA library construction method according to claim 7, characterized in that: The primers used in the PCR of step (21) are a fragment of the reverse transcription primer with a length of 20-30 nt, and a fragment of the TSO primer with a length of 20-30 nt.

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