Link bridge and method for establishing RNA (Ribonucleic Acid) library

Abstract

The invention discloses a connecting bridge and a library building method of an RNA (Ribonucleic Acid) library. The connecting bridge is a double-chain nucleotide fragment consisting of a first chain and a second chain, the first strand sequentially comprises a region A, a region B and a region C from 5 '-3', the region A comprises a desoxyribonucleic acid base and is used for being connected with a joint compound, the region B comprises a desoxyribonucleic acid base and is used for being complementarily paired with the second strand, the region C comprises a ribonucleic acid base and is used for being connected with RNA to be detected, and the second strand sequentially comprises a region D and a region E from 5 '-3' and is used for being complementarily paired with RNA to be detected. The region D comprises a deoxyribonucleic acid base and is used for being complementarily paired with RNA to be detected, and the region E comprises a deoxyribonucleic acid base and is used for being complementarily paired with a first chain. The connecting bridge can be effectively connected with a linker compound at the 5'end of RNA (Ribonucleic Acid).

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a connection bridge and a method for building an RNA library. Background Art

[0002] Nanopore RNA direct sequencing technology can sequence natural RNA, without reverse transcription or PCR amplification, and directly read full-length transcripts without sequencing bias. This technology can retain RNA base modification information and detect single-base modification sites such as m6A, m5C, and pseudouridine on a single RNA molecule; it can accurately analyze variable splicing, alternative polyadenylation (APA), fusion genes, and identify new transcripts; in addition, it can also make relatively accurate estimates of poly(A) length and restore true RNA characteristics.

[0003] The current nanopore RNA direct sequencing technology mainly sequences from the 3'-5' direction. This method depends on the fact that the helicase used in the current nanopore RNA direct sequencing needs to work from 3'-5'. In addition, it is more efficient to connect the adapter complex at the 3' end of RNA compared to the 5' end. However, 3'-5' sequencing sometimes loses the information at the 5' end. Summary of the invention

[0004] In view of the above problems, the present invention provides a connecting bridge, wherein the connecting bridge is a double-stranded nucleotide fragment consisting of a first chain and a second chain.

[0005] The first chain includes region A, region B and region C from 5'-3' in sequence, wherein region A includes deoxyribonucleic acid bases for connecting to the linker complex, region B includes deoxyribonucleic acid bases for complementary pairing with the second chain, and region C includes ribonucleic acid bases for connecting to the RNA to be tested.

[0006] The second chain includes region D and region E in sequence from 5' to 3', wherein region D includes deoxyribonucleic acid bases for complementary pairing with the RNA to be detected, and region E includes deoxyribonucleic acid bases for complementary pairing with the first chain.

[0007] Optionally, according to the above-mentioned connecting bridge, the region A includes 3-5 deoxyribonucleic acid bases, the region B includes 8-20 deoxyribonucleic acid bases, the region C includes 3-6 ribonucleic acid bases, the region D includes 8-15 deoxyribonucleic acid bases, and the region E includes 8-20 deoxyribonucleic acid bases. The region A may also include a spacer, such as at least one of 5SpC3, iSpC3 and iSp18.

[0008] Optionally, according to the above-mentioned connecting bridge, the region A includes deoxyribonucleic acid bases and spacers in sequence from 5' to 3', and the number of the spacers is 1-5.

[0009] Optionally, according to the above-mentioned connecting bridge, the first chain is shown as SEQ ID NO.1 or SEQ ID NO.11, and the second chain is shown as SEQ ID NO.2 or SEQ ID NO.4.

[0010] The present invention also provides a method for constructing an RNA library, comprising: connecting the above-mentioned connection bridge with the RNA to be tested to obtain a connection product; and connecting the connection product with the adapter complex to obtain an RNA library.

[0011] The present invention also provides another method for building an RNA library, comprising: connecting the above-mentioned connecting bridge to the RNA to be tested to obtain a connecting product; reverse transcribing the connecting product to obtain the RNA / DNA hybrid chain; connecting the RNA / DNA hybrid chain to the adapter complex to obtain an RNA library. In this method, the first chain region A of the connecting bridge preferably contains a spacer, which can terminate reverse transcription, prevent the first chain cDNA from extending to the 5' end of the connecting bridge, and ensure the subsequent connection efficiency with the adapter complex.

[0012] Optionally, in the above RNA library construction method, the condition for connecting the connecting bridge to the RNA to be tested is connecting at room temperature for 60 minutes.

[0013] Optionally, in the above RNA library construction method, the ratio of the amount of the first linker chain: the second linker chain: the RNA substance to be detected is 3:3:1.

[0014] The present invention also provides a nanopore library construction kit, comprising the above-mentioned connecting bridge and adapter complex.

[0015] Optionally, the adapter complex comprises a non-RNA polynucleotide sequencing sequence and a helicase. The non-RNA polynucleotide comprises a first section for guiding the fragment to be sequenced into the nanopore, a second section for binding the helicase, a third section for controlling the speed of the helicase moving along the fragment to be sequenced, and a fourth section for connecting the connecting bridge.

[0016] The application of the above-mentioned connecting bridge, the above-mentioned method, or the above-mentioned kit in any of the following also falls within the protection scope of the present invention:

[0017] (1) Nanopore sequencing RNA library construction

[0018] (2) Preparing products for nanopore sequencing RNA library construction;

[0019] (3) Nanopore sequencing;

[0020] (4) Preparing nanopore sequencing products;

[0021] (5) Testing helicase;

[0022] (6) Preparation of test helicase products.

[0023] In order to develop a helicase for nanopore 5'-3' RNA direct sequencing, this patent provides a connection bridge that can effectively connect the adapter complex at the 5' end of RNA, and uses the existing DNA helicase to complete the collection of nanopore RNA 5'-3' signals. This method can be used to test multiple helicases in the future to find a suitable helicase for nanopore 5'-3' RNA direct sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the connection bridge results.

[0025] Figure 2 This is a denaturing polyacrylamide electrophoresis diagram of the determination of the connection efficiency between the connection bridge and the synthetic single-stranded RNA under different connection conditions in Example 1.

[0026] Figure 3 Flow chart for sample ligation efficiency determination.

[0027] Figure 4 This is the electrophoresis diagram of the amplified product for determining the connection efficiency between the connecting bridge and single-stranded RNA of different lengths in Example 1.

[0028] Figure 5 Flow chart of library construction for direct sequencing of single-stranded RNA in the 5′-3′ direction.

[0029] Figure 6 This is the sample pass signal of 40nt RNA library construction in Example 2.

[0030] Figure 7 This is the sample pass signal of 500nt mRNA library construction in Example 2.

[0031] Figure 8 This is the sample pass signal of 1000nt mRNA library construction 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] The schematic diagram of the connecting bridge structure of the present invention is as follows Figure 1 As shown. The connecting bridge is a double-stranded nucleotide fragment composed of a first chain and a second chain. The first chain includes region A, region B and region C from 5'-3' in sequence, region A includes deoxyribonucleic acid bases for connecting the adapter complex, region B includes deoxyribonucleic acid bases for complementary pairing with the second chain, and region C includes ribonucleic acid bases for connecting the RNA to be tested. The second chain includes region D and region E from 5'-3' in sequence, region D includes deoxyribonucleic acid bases for complementary pairing with the RNA to be tested, and region E includes deoxyribonucleic acid bases for complementary pairing with the first chain.

[0035] Example 1: Determination of the efficiency of the connection between the connecting bridge and the single-stranded RNA

[0036] 1. Determination of the ligation efficiency of the ligation bridge and synthetic single-stranded RNA under different ligation conditions

[0037] Design and synthesize bridge primer sequences

[0038] Sequence 1 (i.e. the first strand of the connecting bridge), synthetic sequence, (SEQ ID NO. 1): 5'phos-CACTAGATGTGTATAAGAGACAG, positions 1-17 are DNA, and positions 18-23 are RNA.

[0039] Sequence 2 (i.e. the second strand of the connecting bridge), synthetic DNA sequence (SEQ ID NO. 2): GTCAACCCCTGTCTCTTATACACATCTA.

[0040] Sequence three (i.e., the RNA to be tested), synthetic RNA sequence (SEQ ID NO.3): GGGUUGACCUUUCUCUCCCAUAUUGCAGUCGCGGCACGAU.

[0041] Annealing buffer (50mM HEPES, pH 8.0 and 100mM KCl) was used to dissolve sequence 1, sequence 2 and sequence 3, respectively, to prepare stock solutions 1, 2 and 3 with a concentration of 100μM. Stock solutions 1, 2 and 3 were mixed with annealing buffer (specific amounts are shown in Table 1) at a ratio of 1:1:1, 2:2:1 and 3:3:1, respectively, and annealed. The annealing procedure was: the temperature was reduced from 70°C to 22°C at -2°C / min to obtain annealing complexes of different ratios of three stock solutions 1, 2 and 3. Each ratio of annealing complex was divided into three equal parts, one as a negative control, and the other two were placed at room temperature (20°C) for connection for 30min and 60min to obtain connection products (see Table 2 for the connection system). The connection products were run on denatured polyacrylamide (10% PAGE, 7MUrea) electrophoresis to detect the connection efficiency of the connection bridge and the synthetic single-stranded RNA under different connection conditions.

[0042] Table 1

[0043]

[0044] Table 2

[0045] Components Dosage (μL) Annealing complex 1 NEB T4 ligase(M0202) 1.5 NEB 5×T4 Quick ligation buffer 3 <![CDATA[Nuclease-free H2O]]> 9.5 Total volume 15

[0046] Figure 2 The figure is a denaturing polyacrylamide electrophoresis diagram. Through quantitative analysis of the proportion of single-stranded RNA surplus, the proportion of the connecting bridge to the synthetic single-stranded RNA is 1:1:1, 2:2:1, 3:3:1, and the connection is carried out at room temperature for 30 minutes. The proportion of single-stranded RNA surplus is 46.5%, 13.0%, and 8.0%, respectively; the proportion of the connecting bridge to the synthetic single-stranded RNA is 1:1:1, 2:2:1, 3:3:1, and the connection is carried out at room temperature for 60 minutes. The proportion of single-stranded RNA surplus is 14.0%, 1.7%, and 0.8%, respectively. It is concluded that under the above test conditions, the connection efficiency is best when the proportion of the connecting bridge to the synthetic single-stranded RNA is 3:3:1 and the connection is carried out at room temperature for 60 minutes.

[0047] 2. Determination of the efficiency of connecting bridges to single-stranded RNAs of different lengths

[0048] Figure 3This is a flow chart for determining the sample connection efficiency. As shown in the figure, the connection bridge and ssRNA (single-stranded RNA) are connected to obtain a connection product, which is reverse transcribed using reverse transcriptase to obtain an RNA / DNA hybrid chain (black is DNA, blue is RNA); then the RNA chain in the RNA / DNA hybrid chain is digested with RNase H; the digested product is purified; finally, PCR amplification is performed using two sets of primers A / B, and the PCR product is run on agarose gel and the connection efficiency is quantified using ImageJ. The amount of amplified product of primers ① and primers ③ of primer group A represents the total amount of ssRNA, and the amount of amplified product of primers ② and primers ③ of primer group B represents the amount of ssRNA connected to the connection bridge. The connection efficiency is the percentage of the brightness of the electrophoretic bands of the amplified product of primers of primers of group B and primers of group A.

[0049] 1) Preparation of connecting bridge

[0050] Design and synthesize bridge primer sequences

[0051] Sequence four (i.e. the second strand of the connecting bridge), synthetic DNA sequence, (SEQ ID NO.4): AGTCTCCCCTGTCTCTTATACACATCTA.

[0052] Sequence 11 (i.e., the first strand of the connecting bridge), synthetic sequence, (SEQ ID NO. 11): 5'phos-CACTT / iSp18 / / iSp18 / TAGATGTGTATAAGAGACAG. Positions 1-5 and 8-21 are DNA, positions 6-7 are two iSp18s, i.e., two spacers, and positions 22-27 are RNA.

[0053] Annealing buffer (50mM HEPES, pH 8.0 and 100mM KCl) was used to dissolve sequence 4 and sequence 11, respectively, to prepare stock solutions 4 and 11 with a concentration of 100μM. Stock solutions 1 and 4 were mixed at a ratio of 1:1 to prepare a linker (I) with a final concentration of 10μM, and stock solutions 11 and 4 were mixed at a ratio of 1:1 to prepare a linker (II) with a final concentration of 10μM; both were annealed, and the annealing procedure was: the temperature was reduced from 70℃ to 22℃ at -2℃ / min, and stored in a -20℃ refrigerator for subsequent experiments.

[0054] 2) Preparation of single-stranded RNA of different lengths

[0055] Taking the preparation of 1000nt mRNA as an example, lambda DNA (NEB) was used as a template, and primers 1000nt F (SEQ ID NO.5) and 1000nt R (SEQ ID NO.6) were used as primers to perform PCR amplification to obtain a 1000nt DNA sequence containing a T7 RNA Polymerase binding site. The obtained DNA sequence was purified and used as a template in the subsequent transcription reaction. TM T7 High Yield RNA Synthesis Kit (NEB) was used for transcription reaction to obtain lambda 1000nt mRNA, and the RNA was purified by TRIzol method; DNaseⅠ (GenScript) was used to remove the DNA template in the mRNA, and the mRNA was treated at 37℃ for 30min, and then the RNA was further purified by TRIzol method to obtain 1000nt mRNA.

[0056] Using the same preparation method, 1600nt F primer (SEQ ID NO.7) and 1600nt R primer (SEQ ID NO.8) were used to prepare 1600nt mRNA; 2000nt F primer (SEQ ID NO.9) and 2000nt R primer (SEQ ID NO.10) were used to prepare 2000nt mRNA; and the mRNA was stored in a -80°C refrigerator.

[0057] 3) Connection of the bridge to single-stranded RNA of different lengths

[0058] According to Table 3, each component was added in sequence and mixed, and used for the ligation reaction between the bridge (I) and the single-stranded RNA to obtain a ligation product. The ligation procedure was: 20°C, 1h;

[0059] Table 3

[0060]

[0061]

[0062] 4) Determination of the efficiency of the ligation between the ligation bridge and single-stranded RNA of different lengths

[0063] Using SuperScript TM The ligation product in 4) was reverse transcribed using IV reverse transcriptase (Invitrogen, 18090010) to obtain an RNA / DNA hybrid chain; the RNA chain in the RNA / DNA hybrid chain was then digested using RNase H (NEB M0297s); the digested product was purified using 1.4× magnetic beads (Beckman A63882 AMPure XP); and finally the ligation efficiency was quantified by PCR amplification.

[0064] The 1000nt mRNA magnetic bead purified product was amplified using the primer pair A (1000nt F with sequence 1000nt R) and the primer pair B (primer (SEQ ID NO.14) with 1000nt R); the 1600nt mRNA magnetic bead purified product was amplified using the primer pair A (1600nt F with 1600nt R) and the primer pair B (primer (SEQ ID NO.14) with 1600ntR); the 2000nt mRNA magnetic bead purified product was amplified using the primer pair A (2000nt F with 2000nt R) and the primer pair B (primer (SEQ ID NO.14) with 2000nt R).

[0065] Figure 4 The electrophoresis diagram of the amplified product was used to quantify the ligation efficiency. It was found that the ligation efficiency of the ligation bridge with 1000nt, 1600nt and 2000nt mRNA was no less than 70%, namely 95.3%, 71.7% and 84.3% respectively. The ligation efficiency of 1000nt mRNA was as high as 95%.

[0066] Example 2 Direct sequencing of single-stranded RNA of different lengths in the 5′-3′ direction

[0067] The above embodiment confirmed through experiments that the connection method with single-stranded RNA by means of a connection bridge is feasible, and the optimal connection conditions have been optimized. This embodiment uses this method to detect the current signal of direct sequencing of single-stranded RNA of different lengths in the 5'-3' direction.

[0068] Figure 5 The flowchart of library construction for direct sequencing of single-stranded RNA in the 5'-3' direction. As shown in the figure, there are two methods for library construction. One method for library construction includes connecting the connection bridge to ssRNA to obtain a connection product; connecting the connection product to a linker complex to obtain an RNA library. Another method for library construction includes: connecting the connection bridge to ssRNA to obtain a connection product; reverse transcribing the connection product to obtain an RNA / DNA hybrid chain; connecting the RNA / DNA hybrid chain to a linker complex (i.e., the linker complex in the figure) to obtain an RNA library.

[0069] The specific operations are as follows:

[0070] 1. Connecting bridge to synthetic single-stranded RNA

[0071] The connection method of the synthetic 40nt RNA and the connecting bridge was consistent with the connection method in step 1 of "1. Determination of the connection efficiency of the connecting bridge and the synthetic single-stranded RNA under different connection conditions" in Example 1, and the optimal connection conditions were used: the ratio of the first chain and the second chain of the connecting bridge to the synthetic single-stranded RNA was 3:3:1, and the connection was carried out at room temperature for 60 minutes.

[0072] Using primer 500nt F (SEQ ID NO.12) and primer 500nt R (SEQ ID NO.13), the method in “2. Determination of the ligation efficiency of the linker bridge and single-stranded RNAs of different lengths” of Example 1 was used to prepare 500nt mRNA;

[0073] The connection of 500nt mRNA and 1000nt mRNA (prepared in Example 1) to the connecting bridge is consistent with the connection method of steps 2)-3) of "2. Determination of the connection efficiency of the connecting bridge and single-stranded RNA of different lengths" in Example 1. The 1000nt mRNA is connected to the connecting bridge (I); after the 500nt mRNA is connected to the connecting bridge (II), a reverse transcription primer is used to form an RNA / DNA hybrid chain, and two iSp18s are designed at the 5' end of the connecting bridge (II) to terminate the reverse transcription, prevent the first-chain cDNA from extending to the 5' end of the connecting bridge, and ensure the subsequent connection efficiency with the adapter complex.

[0074] 2. Ligation of adapter complex and purification

[0075] The barcode library construction kit (Cat. No. PY-BLP101, Beijing Puyi Biotechnology Co., Ltd.) was used to connect and purify the sample adapter complex to complete the library construction. The detailed operation is as follows

[0076] 1. Prepare the following reaction system in a 1.5 mL centrifuge tube:

[0077] Table 4

[0078] Components volume Bridge-mRNA 1000ng Ligation Buffer 25μL Adaptor complex 5μL DNA ligase 8μL <![CDATA[Nuclease-free H2O]]> up to100

[0079] After gently pipetting to mix, do not shake, place the centrifuge tube in a 20℃ metal bath and incubate for 15 minutes.

[0080] 2. Add 1.2 times the volume of resuspended RNA magnetic beads into the centrifuge tube and mix well by pipetting, then incubate at room temperature for 10 minutes.

[0081] 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;

[0082] 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;

[0083] 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;

[0084] 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.

[0085] 7. Use Qubit fluorescence quantification instrument to detect library concentration and take 200ng library sample for sequencing.

[0086] 4. RNA sample sequencing

[0087] The library samples were sequenced using the nanopore gene sequencing platform PolyseqOne (Cat. No. PY-NSRO01, Beijing Puyi Biotechnology Co., Ltd.).

[0088] Figure 6 For the synthesis of 40nt RNA library sample pass signal, Figure 7 For the 500nt mRNA library sample pass signal, Figure 8 The sample pass-through signal for 1000nt mRNA library construction. The results show that for RNAs of different lengths, nanopore sequencing signals with obvious steps and smooth and complete signals can be obtained in the experiment; the signal noise can be reduced by screening suitable helicases and nanopore proteins to complete RNA 5′-3′ direction direct sequencing. The collection and data analysis of pass-through signals of RNA samples of different lengths show that this method can realize direct library construction in the 5'-3' direction of single-stranded RNA.

[0089] 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 connecting bridge, wherein the connecting bridge is a double-stranded nucleotide fragment consisting of a first strand and a second strand, characterized in that: The first strand includes region A, region B and region C in order from 5' to 3', wherein region A includes deoxyribonucleic acid bases for connecting to the adapter complex, region B includes deoxyribonucleic acid bases for complementary pairing with the second strand, and region C includes ribonucleic acid bases for connecting to the RNA to be tested; The second strand includes region D and region E in order from 5' to 3', wherein region D includes deoxyribonucleic acid bases for complementary pairing with the RNA to be detected, and region E includes deoxyribonucleic acid bases for complementary pairing with the first strand.

2. The connecting bridge according to claim 1, characterized in that: The region A includes 3-5 deoxyribonucleic acid bases, the region B includes 8-20 deoxyribonucleic acid bases, the region C includes 3-6 ribonucleic acid bases, the region D includes 8-15 deoxyribonucleic acid bases, and the region E includes 8-20 deoxyribonucleic acid bases.

3. The connecting bridge according to claim 1, characterized in that: The region A includes deoxyribonucleic acid bases and spacers in sequence from 5' to 3', and the number of the spacers is 1 to 5.

4. The connecting bridge according to any one of claims 1 to 3, characterized in that: The first chain is shown as SEQ ID NO.1 or SEQ ID NO.11, and the second chain is shown as SEQ ID NO.2 or SEQ ID NO.

4.

5. A method for constructing an RNA library, characterized in that: include: Connect the connection bridge described in any one of claims 1 to 4 to the RNA to be tested to obtain a connection product; and connect the connection product to the adapter complex to obtain an RNA library.

6. A method for constructing an RNA library, characterized in that: include: Connecting the connection bridge described in any one of claims 1 to 4 to the RNA to be tested to obtain a connection product; reverse transcribing the connection product to obtain the RNA / DNA hybrid chain; connecting the RNA / DNA hybrid chain to the adapter complex to obtain an RNA library.

7. A nanopore library construction kit, characterized in that: The method comprises a connecting bridge and a linker complex as claimed in any one of claims 1 to 4.

8. The method according to claim 5 or 6, or the kit according to claim 7, characterized in that: The adaptor complex includes a non-RNA polynucleotide sequencing sequence and a helicase.

9. Use of the linker according to any one of claims 1 to 4, the method according to claim 5 or 6, or the kit according to claim 7 in any of the following: (1) Nanopore sequencing RNA library construction (2) Preparing products for nanopore sequencing RNA library construction; (3) Nanopore sequencing; (4) Preparing nanopore sequencing products; (5) Testing helicase; (6) Prepare the product for testing helicase.

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