ENZYME REACTION SOLUTION FOR CONSTRUCTING SEQUENCING LIBRARY AND USE THEREOF

Abstract

Provided in the present invention is an enzyme reaction solution for constructing a sequencing library and the use thereof. The enzyme reaction solution comprises an enzyme composition and a reaction buffer, wherein the enzyme composition comprises a nucleic acid endonuclease, a DNA polymerase, and a polynucleotide kinase; and the reaction buffer comprises a metal salt, a substrate, and a buffer medium aqueous solution. The present application aims to optimize the formulation of an enzyme reaction solution. The cleavage, terminal repair and addition of A to the terminal of a nucleic acid sample is achieved by a one-step reaction. In a suitable buffer system, the enzyme digestion reaction rate and the terminal repair reaction rate reach a balance. In the case where the initial amount of the sample is 100 pg to 1 .Math.g and the processing time is the same, a sequencing library with a consistent length distribution is obtained.

Claims

1. An enzyme reaction solution for constructing a sequencing library, comprising an enzyme composition and a reaction buffer[;], wherein the enzyme composition comprises an endonuclease, a DNA polymerase, and a polynucleotide kinase; and the reaction buffer comprises a metal salt, a substrate, and an aqueous buffer medium solution.

2. The enzyme reaction solution according to claim 1, wherein the endonuclease comprises any one or a combination of at least two of endonuclease dsDNase, T7 endonuclease, salt-active endonuclease SAN, endonuclease Vvn or endonuclease DNaseI.

3. The enzyme reaction solution according to claim 1, wherein the DNA polymerase comprises a low-temperature DNA polymerase and/or a thermostable DNA polymerase.

4. The enzyme reaction solution according to claim 1, wherein the low-temperature DNA polymerase comprises any one or a combination of at least two of T4 DNA polymerase, T7 DNA polymerase, DNA polymerase I, or the large fragment Klenow of DNA polymerase I.

5. The enzyme reaction solution according to claim 1, wherein the thermostable DNA polymerase comprises Taq DNA polymerase.

6. The enzyme reaction solution according to claim 1, wherein the polynucleotide kinase comprises T4 polynucleotide kinase.

7. The enzyme reaction solution according to claim 1, wherein the enzyme composition further comprises an accessory protein; optionally, the accessory protein comprises a bovine serum albumin and/or a single-chain binding protein; and optionally, the single-chain binding protein comprises an E. coli single-chain binding protein and/or a T4 bacteriophage single-chain binding protein.

8. The enzyme reaction solution according to claim 1, wherein the metal salt comprises a metal cation, comprising any one or a combination of at least two of Mg.sup.2+, Mn.sup.2+, Na.sup.+ or Ca.sup.2+.

9. The enzyme reaction solution according to claim 1, wherein the substrate comprises any one or a combination of at least two of dNTPs, dATP or ATP.

10. The enzyme reaction solution according to claim 1, wherein the buffer medium comprises 4-hydroxyethyl piperazine ethanesulfonic acid and/or tris(hydroxymethyl)aminomethane.

11. The enzyme reaction solution according to claim 1, wherein the final concentration of the endonuclease in the enzyme reaction solution is 0.003 to 0.05 U/.Math.L or 0.1 to 0.5 ng/.Math.L; optionally, the final concentration of the low-temperature DNA polymerase in the enzyme reaction solution is 0.01 to 0.05 U/.Math.L; optionally, the final concentration of the thermostable DNA polymerase in the enzyme reaction solution is 0.03 to 1.2 U/.Math.L; optionally, the final concentration of the polynucleotide kinase in the enzyme reaction solution is 0.05 to 0.2 U/.Math.L; optionally, the final concentration of the accessory protein in the enzyme reaction solution is 0.05 to 1 .Math.g/.Math.L; optionally, the final concentration of the Mg.sup.2+ in the enzyme reaction solution is 0 to 20 mM; optionally, the final concentration of the Mn.sup.2+ in the enzyme reaction solution is 0.05 to 1 mM; optionally, the final concentration of the Na.sup.+ in the enzyme reaction solution is 0 to 50 mM; optionally, the final concentration of the Ca.sup.2+ in the enzyme reaction solution is 0 to 10 mM; optionally, the final concentration of the dNTPs in the enzyme reaction solution is 0.05 to 0.5 mM; optionally, the final concentration of the dATP in the enzyme reaction solution is 0.1 to 2 mM; optionally, the final concentration of the ATP in the enzyme reaction solution is 1 to 10 mM; and optionally, the final concentration of the buffer medium in the enzyme reaction solution is 10 to 50 mM.

12. A method for constructing a sequencing library, comprising: (1) adding a target nucleic acid into the enzyme reaction solution according to claim 1, incubating, and performing fragmentation, end repair, 5′ phosphorylation and 3′ A-tailing of the target nucleic acid; (2) ligating an incubation product with a sequencing adaptor; and (3) performing PCR on a ligation product to obtain a sequencing library.

13. The construction method according to claim 12, wherein the target nucleic acid is added in an amount of 100 pg to 1 .Math.g.

14. The construction method according to claim 12, wherein the incubation conditions involve keeping at 35 to 40° C. for 10 to 20 min, and at 60 to 70° C. for 20 to 40 min.

15. A kit for constructing a sequencing library, comprising the enzyme reaction solution according to claim 1; and optionally, the kit further comprises any one or a combination of at least two of a sequencing adaptor, a ligation reaction reagent or a PCR reagent.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0070] FIG. 1 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 1;

[0071] FIG. 2 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 2;

[0072] FIG. 3 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 3;

[0073] FIG. 4 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 4;

[0074] FIG. 5 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 5;

[0075] FIG. 6 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 6;

[0076] FIG. 7 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 7;

[0077] FIG. 8 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 8;

[0078] FIG. 9 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 9;

[0079] FIG. 10 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 10;

[0080] FIG. 11 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 11;

[0081] FIG. 12 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 12;

[0082] FIG. 13 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 13;

[0083] FIG. 14 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 14;

[0084] FIG. 15 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 15;

[0085] FIG. 16 shows the length distribution of the sequencing library constructed using the enzyme reaction solution of combination 16.

DETAILED DESCRIPTION

[0086] In order to further illustrate the technical means adopted in the present application and its effects, the present application will be further described below with reference to the examples and the drawings. It should be understood that the specific implementations described herein are only used to explain the present application, but not to limit the present application.

[0087] Where no specific technique or condition is indicated in the examples, the procedures should be carried out in accordance with the techniques or conditions described in the literature in the art, or in accordance with the product manuals. The used reagents or instruments without indication of the manufacturers are all conventional products that can be commercially purchased through regular channels.

Example 1. Preparation of Enzyme Reaction Solution

[0088] In this example, an enzyme reaction solution was first prepared, including two portions: the reaction buffer as shown in Table 1 and the enzyme composition as shown in Table 2. The reaction buffer in Table 1 and the enzyme composition in Table 2 were cross-combined to obtain enzyme reaction solutions with different formulations as shown in Table 3. The manufacturer and Cat# of protease are shown in Table 4, and the amino acid sequence of Vibrio vulnificus nuclease Vvn is shown in SEQ ID NO: 1.

TABLE-US-00001 Reaction buffer A B C D Component Concentration Component Concentration Component Concentration Component Concentration HEPES 200 mM HEPES 200 mM Tris 300 mM Tris 300 mM MgCl.sub.2 150 mM MgCl.sub.2 50 mM MgCl.sub.2 150 mM MgCl.sub.2 150 mM CaCl.sub.2 30 mM CaCl.sub.2 30 mM CaCl.sub.2 30 mM CaCl.sub.2 30 mM NaCl 100 mM NaCl 100 mM NaCl 100 mM NaCl 100 mM MnCl.sub.2 5 mM MnCl.sub.2 1 mM MnCl.sub.2 5 mM MnCl.sub.2 1 mM dNTPs 1 mM dNTPs 1 mM dNTPs 1 mM dNTPs 1 mM dATP 6 mM dATP 6 mM dATP 6 mM dATP 6 mM ATP 50 mM ATP 50 mM ATP 50 mM ATP 50 mM

TABLE-US-00002 Enzyme composition Enzyme composition 1 Enzyme composition 2 Enzyme composition 3 Enzyme composition 4 Component Dosage Component Dosage Component Dosage Component Dosage T4 PNK 5U T4 PNK 5U T4 PNK 5U T4 PNK 5U BSA 15 .Math.g BSA 15 .Math.g BSA 15 .Math.g BSA 15 .Math.g T4 GP32 5 .Math.g E.coli SSB 5 .Math.g T4 GP32 5 .Math.g T4 GP32 5 .Math.g Vvn 10 ng dsDNase 0.5 U SAN 0.3 U DNaseI 0.3 U T7 endonuclea se 0.5 U Taq DNA polymerase 1.5 U Taq DNA polymerase 1.5 U Taq DNA polymerase 1.5 U Taq DNA polymerase 1.5 U Klenow 5U Klenow 25 U E. coli DNA polymerase I 3 U T4 DNA polymerase 45 U Glycerin supplement ed to 10 .Math.L Glycerin supplement ed to 10 .Math.L Glycerin supplement ed to 10 .Math.L Glycerin supplement ed to 10 .Math.L

TABLE-US-00003 Enzyme reaction solution Combination No. Reaction buffer + Enzyme composition Combination 1 Reaction buffer A + Enzyme composition 1 Combination 2 Reaction buffer A + Enzyme composition 2 Combination 3 Reaction buffer A + Enzyme composition 3 Combination 4 Reaction buffer A + Enzyme composition 4 Combination 5 Reaction buffer B + Enzyme composition 1 Combination 6 Reaction buffer B + Enzyme composition 2 Combination 7 Reaction buffer B + Enzyme composition 3 Combination 8 Reaction buffer B + Enzyme composition 4 Combination 9 Reaction buffer C + Enzyme composition 1 Combination 10 Reaction buffer C + Enzyme composition 2 Combination 11 Reaction buffer C + Enzyme composition 3 Combination 12 Reaction buffer C + Enzyme composition 4 Combination 13 Reaction buffer D + Enzyme composition 1 Combination 14 Reaction buffer D + Enzyme composition 2 Combination 15 Reaction buffer D + Enzyme composition 3 Combination 16 Reaction buffer D + Enzyme composition 4

TABLE-US-00004 Manufacturer and Cat# of protein/enzyme Name of protein/enzyme Manufacturer Cat# T4 PNK Vazyme N102-01 BSA Sigma V900933 T4 GP32 NEB M0300L Vvn In-house expressed See the sequence below T7 Endonuclease Vazyme EN303 Taq DNA polymerase Vazyme P101 Klenow Vazyme N104 dsDNase thermo EN0771 SAN arcticzymes 70910 DNaseI Vazyme EN401 E.ColI SSB abcam ab123224

[0089] Amino acid sequence of Vibrio vulnificus nuclease Vvn (SEQ ID NO: 1):

[0090] Ala Pro Pro Ser Thr Phe Ser Ala Ala Lys Gln Gln Ala Ala Lys Ile Tyr Gln Asp His Pro Ile Thr Phe Tyr Cys Gly Cys Asp Ile Glu Trp Gln Gly Lys Lys Gly Ile Pro Asn Leu Glu Thr Cys Gly Tyr Gln Val Arg Lys Ser Gln Thr Arg Ala Ser Arg Ile Glu Trp Glu His Val Val Pro Ala Trp Gln Phe Gly His His Arg Gln Cys Trp Gln Lys Gly Gly Arg Lys Asn Cys Ser Lys Asn Asp Gln Gln Phe Arg Leu Met Glu Ala Asp Leu His Asn Leu Ser Pro Ala Ile Gly Glu Val Asn Gly Asp Arg Ser Asn Phe Asn Phe Ser Gln Trp Asn Gly Val Asp Gly Val Ser Tyr Gly Arg Cys Glu Met Gln Val Asn Phe Lys Gln Arg Lys Val Met Pro Gln Thr Glu Leu Arg Gly Ser Ile Ala Arg Thr Tyr Leu Tyr Met Ser Gln Glu Tyr Gly Phe Gln Leu Thr Lys Gln Gln Gln Leu Met Gln Ala Trp Asn Lys Ser Tyr Pro Val Asp Glu Trp Glu Cys Ser Arg Asp Asp Arg Ile Ala Lys Ile Gln Gly Asn His Asn Pro Phe Val Gln Gln Ser Cys Gln Thr Gln.

Example 2. Fragmentation and End Repair of a DNA Template

[0091] In this example, salmon sperm gDNA was used as a template, and the initial inputs were 100 pg, 1 ng, 10 ng, 100 ng and 1 .Math.g, respectively. gDNA was subjected to fragmentation, end repair, 3′ A-tailing, and 5′ phosphorylation using the different enzyme reaction solutions of Example 1, and in subsequent examples, the adaptor ligation reactions were performed using Rapid DNA Ligation Buffer and Rapid DNA Ligase in the Vayzme#ND607 VAHTS® Universal DNA Library Prep Kit for Illumina® V3, the adaptor ligation products and PCR amplification products were purified using Vazyme#N401 VAHTS® DNA Clean Beads, and the purified ligation products were amplified and enriched using VAHTS HiFi Amplification Mix and PCR Primer Mix 3 for Illumina in the Vayzme#ND607 VAHTS® Universal DNA Library Prep Kit for Illumina® V3, wherein the adaptors used for ligation reactions were VAHTS® DNA Adapters for Illumina® (Vazyme #N801).

[0092] The fragmentation, end repair, 5′ phosphorylation and 3′ dA-tailing reaction system for the target DNA is shown in Table 5, wherein the formulation of an enzyme composition adopted one of the combinations 1 to 16 in Example 1, the input X .Math.L of target DNA corresponded to 100 pg, 1 ng, 10 ng, 100 ng and 1 .Math.g, respectively, the reactions conditions involved incubating at 37° C. for 15 min, and at 65° C. for 30 min, and the resulting fragmented products were subjected to sequencing library construction.

TABLE-US-00005 Fragmentation reaction system Component Dosage Reaction buffer 5 .Math.L Enzyme composition 10 .Math.L Target DNA X .Math.L Sterilized ddH.sub.2O Made up to 50 .Math.L

Example 3. Construction of a Sequencing Library

[0093] In this example, the fragmented products prepared in Example 2 were subjected to adaptor ligation reactions, PCR amplification reactions and purification to construct a sequencing library. The adaptor ligation reaction system is shown in Table 6, and the formulated system was incubated at 20° C. for 15 min for adaptor ligation; and 60 .Math.L of VAHTS DNA Clean Beads were used to purify the ligation product, and the elution volume was 20/22.5 .Math.L.

[0094] The purified product was subjected to PCR amplification, the system is shown in Table 7, and the conditions are shown in Table 8; and 45 .Math.L of VAHTS DNA Clean Beads were used to purify the amplification product, and the elution volume was 20/22.5 .Math.L.

TABLE-US-00006 Adaptor ligation reaction system Component Volume Fragmented purified product 50 .Math.L Ligation reaction buffer (Rapid DNA Ligation Buffer) 25 .Math.L DNA ligase (Rapid DNA Ligase) 5 .Math.L Adaptor (DNA Adaptor) 5 .Math.L Sterilized ddH.sub.2O 15 .Math.L

TABLE-US-00007 PCR system Component Volume Purified adaptor ligation product 20 .Math.L Enzyme and its buffer for PCR amplification (VAHTS HiFi Amplification Mix) 25 .Math.L Primer (PCR Primer Mix 3 for Illumina) 5 .Math.L

TABLE-US-00008 PCR reaction procedure Temperature Time 95° C. 3 min 98° C. 20 sec 60° C. 15 sec 72° C. 30 sec 72° C. 5 min 4° C. Preservation

[0095] The adaptor dilution folds and amplification cycle numbers corresponding to different initial DNA inputs are shown in Table 9.

TABLE-US-00009 Adaptor dilution folds and amplification cycle numbers corresponding to different initial DNA inputs gDNA input Adaptor dilution fold Amplification cycle numbers 100 pg 200 folds 14 1 ng 100 folds 12 10 ng 10 folds 8 100 ng Stock solution 3 1 .Math.g Stock solution 2

[0096] An Agilent 2100 DNA1000 chip was used to detect the length distribution of the DNA library. The results are shown in FIG. 1 to FIG. 16. It can be seen that each group of enzyme reaction solutions can achieve effective cleavage, end repair, 5′ phosphorylation modification, and 3′ dA-tailing to DNAs with different inputs within the same treatment time.

[0097] As shown in FIG. 16, the enzyme reaction solution of combination 16 has the best effect, and is fully compatible with the initial DNA input of 100 pg to 1 .Math.g; the distribution range of library fragment lengths has good consistency, mainly in the range of 400 to 500 bp, indicating that when the enzyme composition comprises 0.3 U DNaseI, 45 U T4 DNA polymerase, 1.5 U Taq DNA polymerase, 5 U T4 PNK, 15 .Math.g BSA, and 5 .Math.g T4 GP32, it cooperates with the reaction buffer (300 mM Tris, 150 mM MgCh, 30 mM CaCl.sub.2, 100 mM NaCl, 1 mM MnCl.sub.2, 1 mM dNTPs, 6 mM dATP, and 50 mM ATP) to perfectly balance the enzyme digestion reaction rate and the end repair reaction rate, thereby preparing a sequencing library with a consistent fragment length.

[0098] Furthermore, as shown in FIG. 8, FIG. 11 and FIG. 4, the enzyme reaction solutions of combination 8, combination 11 and combination 4 also have good effects, and have good compatibility with DNAs with an initial input of 100 pg to 1 .Math.g, and the lengths of the prepared fragments are substantially consistent, indicating that when the enzyme composition comprises 0.004 to 0.008 U/.Math.L endonuclease, 0.05 to 0.9 U/.Math.L DNA polymerase, 0.01 to 0.05 U/.Math.L Taq DNA polymerase, 0.05 to 0.3 U/.Math.L T4 PNK, and a 0.2 to 0.6 .Math.g/.Math.L accessory protein, it cooperates with the reaction buffer (100 to 400 mM buffer medium, 100 to 300 mM Mg.sup.2+, 1 to 6 mM Mn.sup.2+, 50 to 250 mM Na.sup.+, 5 to 50 mM Ca.sup.2+, 0.5 to 5 mM dNTPs, 1 to 10 mM dATP, and 20 to 40 mM ATP) to regulate the enzyme digestion reaction rate and the end repair reaction rate, so that the fragment lengths of the prepared sequencing library are relatively consistent.

[0099] As can be seen from FIG. 1, the suitable initial input of the enzyme reaction solution of combination 1 is 100 pg to 10 ng, and further increase of the initial input will affect the fragment length; as can be seen from FIG. 2, the suitable initial input of the enzyme reaction solution of combination 2 is 100 ng to 1 .Math.g; as can be seen from FIG. 3, the suitable initial input of the enzyme reaction solution of combination 3 is 1 ng to 10 ng; as can be seen from FIG. 5, the suitable initial input of the enzyme reaction solution of combination 5 is 100 pg to 100 ng; as can be seen from

[0100] FIG. 6, the suitable initial input of the enzyme reaction solution of combination 6 is 10 ng to 100 ng; as can be seen from FIG. 7, the suitable initial input of the enzyme reaction solution of combination 7 is 100 pg to 10 ng; as can be seen from FIG. 9, the suitable initial input of the enzyme reaction solution of combination 9 is 100 pg to 10 ng; as can be seen from FIG. 10, the suitable initial input of the enzyme reaction solution of combination 10 is 100 pg to 10 ng; as can be seen from FIG. 12, the suitable initial input of the enzyme reaction solution of combination 12 is 1 ng to 100 ng; as can be seen from FIG. 13, the suitable initial input of the enzyme reaction solution of combination 13 is 1 ng to 100 ng; and as can be seen from FIG. 15, the suitable initial input of the enzyme reaction solution of combination 15 is 10 ng to 100 ng.

[0101] In summary, by optimizing the formulation of the enzyme reaction solution, through the mutual cooperation of the enzyme composition and the reaction buffer, the present application optimizes the enzyme digestion reaction rate and the end repair reaction rate in the library construction process, achieves the technical effect of obtaining a sequencing library of a consistent length under the condition of different initial amounts of samples and the same treatment time, and has wide applicability and convenient operability.

[0102] The applicant declares that the present application illustrates the detailed method of the present application through the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the present application must rely on the above-mentioned detailed method for implementation. Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of each raw material for producing the product of the present application and the addition of auxiliary components, the selection of specific methods, and the like, all fall within the scope of protection and disclosure of the present application.