An enzyme composition for low bias DNA fragmentation and a reaction solution thereof

Abstract

The application discloses an enzyme composition for low-preference DNA fragmentation and a reaction solution thereof. The enzyme composition comprises an endonuclease, a nucleotide kinase and a DNA polymerase, the endonuclease comprises any one of DNase I, heat-stable high-salt-tolerant endonuclease HL-SAN or endonuclease IV or a combination of at least two of them, the nucleotide kinase comprises T4 polynucleotide kinase, and the DNA polymerase comprises any one of Taq DNA polymerase, a Klenow fragment of DNA polymerase I or T4 DNA polymerase or a combination of at least two of them. The application develops a one-tube method for genome fragmentation, end repair and A-tailing module, including an enzyme composition and a reaction solution thereof. In view of the problems and deficiencies of the current enzyme library construction, the components are optimized and explored, and a suitable connection module is combined, so that the finally obtained product has low preference, the output library has higher uniformity, and has a wide application prospect in second-generation sequencing.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an enzyme composition for low-bias DNA fragmentation and its reaction solution. Background Technology

[0002] In recent decades, DNA sequencing technology has been applied to molecular biology research, greatly advancing the field's rapid development. While first-generation DNA sequencing technologies, with read lengths up to 1000 bp and accuracy of 99.99%, have helped researchers complete a large amount of sequencing work, their slow speed, high cost, and low throughput have made it difficult to support the growing demand for sequencing. Second-generation sequencing (NGS), also known as high-throughput sequencing (HTS), has been increasingly widely used in scientific research and clinical practice due to its advantages such as low cost and high throughput.

[0003] The main workflow of next-generation sequencing (NGS) consists of three parts: library construction, gene sequencing, and bioinformatics analysis. Library construction, as the initial step in NGS sequencing, significantly impacts the final sequencing results; therefore, ensuring high-quality library construction is crucial. Due to the short read lengths of NGS, the first step in DNA library construction is metagenomic fragmentation. Currently, mechanical and enzymatic fragmentation methods can be used to break down genomic DNA. Compared to the high equipment requirements and long processing time of traditional mechanical fragmentation methods, enzymatic fragmentation is increasingly widely used due to its ease of operation, shorter processing time, and lower cost.

[0004] However, compared to the unbiased random cutting of traditional mechanical fragmentation methods, fragmented enzymatic fragmentation selectively chooses restriction sites on the DNA double strand for cutting. This leads to a decrease in the abundance and diversity of random fragments in the library, resulting in poor library uniformity. This can cause significant deviations in sequencing depth across different regions of the gene, making it difficult to detect some low-abundance gene sites during sequencing, potentially leading to missed detections. Increasing sequencing depth can reduce the number of missed detection sites, but this results in a significant waste of data.

[0005] In summary, current fragmentation enzyme methods suffer from low abundance and diversity of random fragments in the library, poor library uniformity, significant sequencing depth discrepancies in different gene regions, and difficulty in detecting some low-abundance gene loci during sequencing, leading to missed detections. Providing an enzyme composition and reaction solution for DNA fragmentation, suitable for one-tube genome fragmentation, end repair, and A-tailing module addition, to obtain libraries with low bias and higher uniformity has become one of the urgent problems to be solved in the field of biotechnology. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an enzyme composition and reaction solution for low-bias DNA fragmentation. This solves the problems of low abundance and diversity of random fragments in libraries, poor library uniformity, significant deviations in sequencing depth across different gene regions, and difficulty in detecting some low-abundance gene loci during sequencing, leading to missed detections. The enzyme composition and reaction solution provided by this invention have good versatility and are suitable for various types of next-generation sequencing reactions.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an enzyme composition for low-bias DNA fragmentation, the enzyme composition comprising a nuclease, a nucleotide kinase, and a DNA polymerase, wherein the nuclease comprises any one or a combination of at least two of DNase I, the thermo-instable, high-salt-resistant nuclease HL-SAN, or nuclease IV, the nucleotide kinase comprises T4 polynucleotide kinase, and the DNA polymerase comprises any one or a combination of at least two of Taq DNA polymerase, the Klenow fragment of DNA polymerase I, or T4 DNA polymerase.

[0009] This invention develops a module suitable for one-tube genome fragmentation, end repair, 5' phosphorylation, and 3' A-tailing, including an enzyme composition and its reaction solution. It can reduce the bias of DNA fragmentation and address the problems and shortcomings of current enzymatic library construction. Through optimization of each component and matching with a suitable ligation module, the final product has low bias and produces libraries with higher uniformity, showing broad application prospects in next-generation sequencing.

[0010] Preferably, the enzyme composition comprises any one of the following compositions:

[0011] (1) A combination of DNase I, Taq DNA polymerase, Klenow fragment of DNA polymerase I and T4 polynucleotide kinase;

[0012] (2) A combination of heat-instable, high-salt-resistant endonuclease HL-SAN, Taq DNA polymerase, Klenow fragment of DNA polymerase I, and T4 polynucleotide kinase;

[0013] (3) A combination of DNase I, endonuclease IV, Taq DNA polymerase, Klenow fragment of DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase.

[0014] Preferably, the concentration of DNase I in the enzyme composition is 0.1-3 U / μL.

[0015] The specific point values ​​in 0.1-3 above can be 0.1, 0.5, 1, 1.5, 2, 2.5, 3, etc.

[0016] Preferably, the concentration of the heat-instantaneous, high-salt-resistant endonuclease HL-SAN in the enzyme composition is 0.01-3 U / μL.

[0017] The specific point values ​​in the range 0.01-3 can be selected from 0.01, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.9, 2, 2.2, 2.5, 2.7, 2.8, 3, etc.

[0018] Preferably, the concentration of endonuclease IV in the enzyme composition is 0.005-2 U / μL.

[0019] The specific point values ​​in 0.005-2 above can be selected from 0.005, 0.008, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.7, 1.8, 2, etc.

[0020] Preferably, the concentration of Taq DNA polymerase in the enzyme composition is 2-35 U / μL.

[0021] The specific point values ​​in 2-35 above can be selected as 2, 6, 9, 12, 20, 25, 29, 30, 31, 32, 33, 34, 35, etc.

[0022] Preferably, the concentration of the Klenow fragment of DNA polymerase I in the enzyme composition is 0.01-7.8 U / μL.

[0023] The specific point values ​​in the range of 0.01-7.8 can be selected from 0.01, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1.7, 2.8, 3.9, 4, 5, 6, 7, 7.8, etc.

[0024] Preferably, the concentration of T4 DNA polymerase in the enzyme composition is 0.3-40 U / μL.

[0025] The specific point values ​​in the range of 0.3-40 can be selected from 0.3, 0.5, 1, 2, 3, 8, 10, 20, 30, 32, 33, 34, 36, 38, 39, 40, etc.

[0026] Preferably, the concentration of T4 polynucleotide kinase in the enzyme composition is 0.1-3 U / μL.

[0027] The specific point values ​​in 0.1-3 above can be selected from 0.2, 0.5, 1, 1.5, 2.5, etc.

[0028] In a second aspect, the present invention provides a reaction solution of an enzyme composition, the reaction solution comprising: disaccharide and / or disaccharide derivative.

[0029] The disaccharide or disaccharide derivative in the reaction solution of this invention can maintain the structural stability of the protein, enhance the ability of the enzyme's protein functional domain to non-specifically recognize and cleave sites on the DNA double strand, promote the synergistic effect between enzyme compositions, and further reduce the bias of library generation.

[0030] Preferably, the disaccharide comprises sucrose.

[0031] Preferably, the disaccharide derivative includes stachyose.

[0032] Preferably, the concentration of sucrose in the reaction solution is 50-120 mM.

[0033] The specific point values ​​in the range of 50-120 can be selected as 50, 60, 70, 80, 90, 100, 110, 120, etc.

[0034] Preferably, the concentration of stachyose in the reaction solution is 30-80 mM.

[0035] The specific point values ​​in the range of 30-80 can be selected as 30, 40, 50, 60, 70, 80, etc.

[0036] Preferably, the reaction solution further includes a buffer solution, metal salt ions, a detergent, ATP, dATP, and dNTPs.

[0037] Preferably, the buffer solution comprises any one or a combination of at least two of tris(hydroxymethyl)aminomethane acetate, tris(hydroxymethyl)aminomethane hydrochloride, or 4-hydroxyethylpiperazine ethanesulfonic acid.

[0038] Preferably, the reaction solution contains 50-200 mM of tris(hydroxymethyl)aminomethane acetate and has a pH of 7-8.5.

[0039] The specific point values ​​in the range of 50-200 can be selected as 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 190, 200, etc.

[0040] Preferably, the reaction solution contains 100-150 mM of tris(hydroxymethyl)aminomethane hydrochloride and has a pH of 7-9.

[0041] The specific point values ​​in 7-9 above can be 7, 8, 9, etc.

[0042] The specific point values ​​in the range of 100-150 can be selected as 100, 110, 120, 130, 140, 150, etc.

[0043] Preferably, the content of 4-hydroxyethylpiperazine ethanesulfonic acid in the reaction solution is 100-180 mM, and the pH is 7-9.

[0044] The specific point values ​​in 7-9 above can be 7, 8, 9, etc.

[0045] The specific point values ​​in the range of 100-180 can be selected as 100, 110, 120, 130, 140, 150, 160, 180, etc.

[0046] Preferably, the metal salt ion includes any one or a combination of at least two of CaCl2, MgCl2, ZnCl2, NaCl, or KCl.

[0047] Preferably, the CaCl2 content in the reaction solution is 15-50 mM.

[0048] The specific point values ​​in the range of 20-50 can be selected as 15, 30, 40, 50, etc.

[0049] Preferably, the MgCl2 content in the reaction solution is 35-200 mM.

[0050] The specific point values ​​in the range of 50-200 can be selected as 35, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 190, 200, etc.

[0051] Preferably, the ZnCl2 content in the reaction solution is 20-80 mM.

[0052] The specific point values ​​in the range of 20-80 can be selected as 20, 30, 40, 50, 60, 70, 80, etc.

[0053] Preferably, the NaCl content in the reaction solution is 20-200 mM.

[0054] The specific point values ​​in the range of 20-200 can be selected as 20, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 190, 200, etc.

[0055] Preferably, the KCl content in the reaction solution is 30-80 mM.

[0056] The specific point values ​​in the range of 30-80 can be selected as 30, 40, 50, 60, 70, 80, etc.

[0057] Preferably, the detergent comprises any one or a combination of at least two of DTT, Triton X-100, Tween-20 or NP-40.

[0058] Preferably, the DTT content in the reaction solution is 10-50 mM.

[0059] The specific point values ​​in the range of 10-50 can be selected as 10, 20, 30, 40, 50, etc.

[0060] Preferably, the Triton X-100 content in the reaction solution is 0.1-1%.

[0061] The specific point values ​​in the range 0.1-1 above can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0062] Preferably, the content of Tween-20 in the reaction solution is 0.01-1%.

[0063] The specific point values ​​in the range 0.01-1 above can be 0.01, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0064] Preferably, the NP-40 content in the reaction solution is 0.01-1%.

[0065] The specific point values ​​in the range 0.01-1 above can be 0.01, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0066] Preferably, the dATP content in the reaction solution is 0.2-8 mM, and more preferably 2-4 mM.

[0067] The specific point values ​​in the range 0.2-8 above can be 0.2, 0.3, 0.4, 1, 2, 3, 4, 5, 6, 7, 8, etc.

[0068] The specific point values ​​in 2-4 above can be 2, 3, 4, etc.

[0069] The dNTP content in the reaction solution is 0.02-2 mM, preferably 0.02-1 mM.

[0070] The specific point values ​​in 0.02-2 above can be selected from 0.02, 0.03, 0.4, 0.8, 1, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.

[0071] The ATP content in the reaction solution is 5-15 mM.

[0072] The specific point values ​​in 5-10 above can be selected as 5, 6, 7, 8, 9, 10, 11, 15, etc.

[0073] Thirdly, the present invention provides a method for constructing a sequencing library, the method comprising: adding a target nucleic acid to a mixture of an enzyme composition as described in the first aspect and a reaction solution as described in the second aspect, incubating, and performing fragmentation, end repair, 5' phosphorylation, and 3' A-tailing of the target nucleic acid.

[0074] Fourthly, the present invention provides a kit for constructing sequencing libraries, the kit comprising the enzyme composition described in the first aspect or the reaction solution described in the second aspect.

[0075] Preferably, the kit further includes sequencing adapters and ligation reaction reagents.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] (1) The one-tube genome fragmentation, end repair and A-tailing module developed in this invention includes an enzyme composition and its reaction solution. In response to the problems and shortcomings of the current enzymatic database construction, the components are optimized and explored, and used in conjunction with the ligation module. The second-generation sequencing process is completed through the DNA sample to be tested. The effect of the enzyme composition and its reaction solution provided by this invention is verified from the final sequencing results, which further confirms its practical application value in the production of second-generation sequencing.

[0078] (2) The solution provided by this invention has good versatility and is applicable to various types of next-generation sequencing reactions. In the construction of PCR-free libraries, the target gene undergoes two steps: fragmentation, end repair, and A-addition and adapter ligation to obtain a library that has not been amplified by PCR. The advantage of constructing PCR-free libraries for sequencing is that it is faster and more efficient, and it can avoid problems such as poor fidelity caused by PCR amplification. The enzyme composition and reaction solution provided by this invention solve the problems of high restriction site bias and high GC bias under high input levels in the process of PCR-free library construction. Attached Figure Description

[0079] Figure 1 This is a base distribution diagram of the reads from the enzyme digestion library construction and sequencing in Example 1;

[0080] Figure 2 This is a base distribution diagram of the reads from the enzymatic digestion library construction and sequencing in Example 2;

[0081] Figure 3 This is a base distribution diagram of the reads from the enzyme digestion library construction and sequencing in Example 3;

[0082] Figure 4 This is a base distribution diagram of the reads from the enzyme digestion library construction and sequencing in Example 4;

[0083] Figure 5 This is a base distribution diagram of the sequencing reads from the enzymatic digestion library in Example 5.

[0084] Figure 6 This is a base distribution diagram of the reads from the enzymatic digestion library construction and sequencing in Example 6;

[0085] Figure 7 Base distribution diagram of reads from 12619 enzyme digestion library construction and sequencing;

[0086] Figure 8 This is a distribution map of the coverage of different GC regions during enzymatic digestion and sequencing in Example 1.

[0087] Figure 9 This is a diagram showing the coverage distribution of different GC regions during enzymatic digestion and sequencing in Example 2.

[0088] Figure 10 This is a diagram showing the coverage distribution of different GC regions during enzymatic digestion and sequencing in Example 3.

[0089] Figure 11 This is a diagram showing the coverage distribution of different GC regions during sequencing after enzyme digestion in Example 4.

[0090] Figure 12 This is a diagram showing the coverage distribution of different GC regions during sequencing after enzyme digestion in Example 5.

[0091] Figure 13 This is a diagram showing the coverage distribution of different GC regions during sequencing after enzyme digestion in Example 6.

[0092] Figure 14 The distribution of GC region coverage for sequencing of 12619 enzyme digestion library. Detailed Implementation

[0093] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0094] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0095] The sources of proteases involved in the examples are shown in Table 1.

[0096] Table 1

[0097]

[0098] Example 1

[0099] This embodiment provides an enzyme composition and its reaction solution: DNase I 1.5 U / μL, Taq DNA polymerase 1 U / μL, Klenow fragment of DNA polymerase I 2 U / μL, T4 polynucleotide kinase 1 U / μL, reaction solution: Tris-Acetate 75 mM pH 7.5, CaCl2 20 mM, MgCl2 45 mM, NaCl 20 mM, ZnCl2 50 mM, Triton X-100 0.1%, Tween-20 0.01%, dATP 2 mM, dNTP 0.2 mM, ATP 9 mM.

[0100] Example 2

[0101] This embodiment provides an enzyme composition and its reaction solution: 0.5 U / μL of thermostable, high-salt-resistant endonuclease HL-SAN, 5 U / μL of Taq DNA polymerase, 2 U / μL of the Klenow fragment of DNA polymerase I, and 1 U / μL of T4 polynucleotide kinase; the reaction solution consists of: 120 mM HEPES (pH 7.5), 35 mM CaCl2, 35 mM MgCl2, 30 mM NaCl, 60 mM KCl, 0.1% Triton X-100, 0.05% Tween-20, 0.05% NP-40, 2 mM dATP, 0.2 mM dNTP, and 15 mM ATP.

[0102] Example 3

[0103] This embodiment provides an enzyme composition and its reaction solution: DNase I 1 U / μL, endonuclease IV 0.05 U / μL, Taq DNA polymerase 9 U / μL, Klenow fragment of DNA polymerase I 2 U / μL, T4 DNA polymerase 0.5 U / μL, T4 polynucleotide kinase 2 U / μL; reaction solution: Tris-HCl 100 mM pH 7.5, CaCl2 15 mM, MgCl2 50 mM, NaCl 30 mM, NP-40 0.02%, Tween-20 0.01%, dATP 2 mM, dNTP 0.2 mM, ATP 9 mM.

[0104] Example 4

[0105] This embodiment provides an enzyme composition and its reaction solution: DNase I 1.5 U / μL, Taq DNA polymerase 1 U / μL, Klenow fragment of DNA polymerase I 2 U / μL, T4 polynucleotide kinase 1 U / μL; reaction solution: sucrose 50 mM, Tris-Acetate 75 mM pH 7.5, CaCl2 20 mM, MgCl2 45 mM, NaCl 20 mM, ZnCl2 50 mM, Triton X-100 0.1%, Tween-20 0.01%, dATP 2 mM, dNTP 0.2 mM, ATP 9 mM. The only difference between this embodiment and Example 1 is that 50 mM sucrose is added to the reaction solution.

[0106] Example 5

[0107] This embodiment provides an enzyme composition and its reaction solution: HL-SAN 0.5 U / μL (thermally unstable, high-salt resistant endonuclease), 5 U / μL (Taq DNA polymerase), 2 U / μL (Klenow fragment of DNA polymerase I), and 2 U / μL (T4 polynucleotide kinase); Reaction solution: 120 mM sucrose, 120 mM HEPES (pH 7.5), 35 mM CaCl2, 35 mM MgCl2, 30 mM NaCl, 60 mM KCl, 0.1% Triton X-100, 0.05% Tween-20, 0.05% NP-40, 2 mM dATP, 0.2 mM dNTP, and 15 mM ATP. The only difference between this embodiment and Example 2 is the addition of 120 mM sucrose to the reaction solution.

[0108] Example 6

[0109] DNase I 1 U / μL, endonuclease IV 0.05 U / μL, Taq DNA polymerase 9 U / μL, Klenow fragment of DNA polymerase I 2 U / μL, T4 DNA polymerase 0.5 U / μL, T4 polynucleotide kinase 12 U / μL; Reaction solution: stachyose 30 mM, Tris-HCl 100 mM pH 7.5, CaCl2 15 mM, MgCl2 50 mM, NaCl 30 mM, NP-40 0.02%, Tween-20 0.01%, dATP 2 mM, dNTP 0.2 mM, ATP 9 mM. The only difference between this example and Example 3 is that 30 mM stachyose was added to the reaction solution.

[0110] Example 7

[0111] Construct PCR-free sequencing libraries.

[0112] This example uses human female gDNA as a template, with an initial input of 1000 ng. Different enzyme compositions and their corresponding reaction solutions from Examples 1-6 were used to fragment the gDNA template, perform end repair, 5' phosphorylation, and 3' A-tailing, in conjunction with Yisheng Biotechnology. The Rapid DNA Ligation Module for kit (#13580) and BGI Genomics MGIEasyPFAdapters (#1000013460) were used for adapter ligation reactions. Smarter DNAClean Beads (#12600) are used to purify and sort adapter ligation products to obtain PCR-free libraries suitable for sequencing.

[0113] This example also uses commercially available Yisheng Biotechnology. Compare with OnePot Pro DNA FragmentationReagent (#12619). This agent fragments the gDNA template, repairs its ends, phosphorylates it at 5', and adds an A-tail at 3', then is used in conjunction with Yisheng Biotechnology. The adapter ligation reaction was performed using the Rapid DNA Ligation Module for kit (#13580) and BGI Genomics MGIEasyPF Adapters (#1000013460), and Yisheng Biotechnology was used. The Smarter DNAClean Beads (#12600) are used to purify and sort adapter ligation products to construct PCR-free libraries. Refer to the instruction manual for specific operating procedures.

[0114] The target DNA fragmentation / end repair / 5' phosphorylation / 3' A-tailing reaction system is shown in Table 2. The enzyme composition formulations are those of Examples 1-6, and the reaction conditions are 30°C for 15 min and 65°C for 20 min.

[0115] Table 2

[0116] Components Dosage target DNA 1000ng Enzyme composition 5μL reaction solution 10μL 1×TE Fill to 60μL

[0117] The obtained fragmented / terminal repair / 5' phosphorylation / 3' A-tailed product was directly subjected to a linker ligation reaction. The reaction system is shown in Table 3. The reaction conditions were 20℃ incubation for 15 min.

[0118] Table 3

[0119] Components Dosage Fragmentation / Terminal repair / 5' phosphorylation / 3' A-tail addition 60μL Ligation Enhancer 30μL MGIEasy PF Adapters 5μL Ligase (Rapid T4 DNA Ligase 2.0) 5μL

[0120] The joint ligation product obtained after incubation is used The DNA was sorted using Smarter DNA Clean Beads at a sorting ratio of 0.7 / 0.2×, eluted with 22 μL of sterile ddH2O, and 20 μL was recovered to obtain a PCR-free library ready for sequencing. Sequencing was performed using the MGI DNBSEQ-T7 platform in PE150 mode.

[0121] Sequencing data were analyzed. FastP 0.23.1 was used to filter the raw data for adapter sequences and low-quality reads, resulting in high-quality sequencing data (Clean Reads). The same amount of Clean Reads was taken from each sample. FastQC 0.11.9 was used to analyze the selected Clean Reads. The magnitude of base variation corresponding to the Perbase sequencecontent index in the analysis results was used as an indicator of enzyme digestion bias; greater variation indicated a more severe enzyme digestion bias. Experimental analysis results are as follows: Figure 1-7 As shown.

[0122] The selected Clean Reads were aligned with the hg38 reference genome using bwa0.7.17. Samtools 1.16.1 was used to sort the files. The Collect GC Bias Metrics module of picard.jar 1.86.0 was used to analyze the coverage information of different GC regions of the genome. Based on the displayed GC region coverage results, the restriction enzyme's fragmentation preference for regions with different GC contents was determined. The experimental analysis results are as follows: Figure 8-14 As shown.

[0123] Result: From Figure 7 It is known that the next holy creature OnePot Pro DNA Fragmentation Reagent (#12619) has a base fluctuation value of 4-50% at the restriction site; in this invention, the base fluctuation value at the restriction site in Example 1 is 12-42%. Figure 1 ), corresponding to the base fluctuation value of the enzyme cleavage site in Example 4 with added disaccharide was 16%-38% ( Figure 4 Example 2: Base fluctuation at the enzyme cleavage site was 16%-40%. Figure 2 The base fluctuation value of the enzyme cleavage site in Example 5, corresponding to the addition of disaccharides, was 18%-39%. Figure 5 Example 3: Base fluctuation at the enzyme cleavage site was 15%-40%. Figure 3 The base fluctuation value of the enzyme cleavage site in Example 6, corresponding to the addition of disaccharide derivatives, was 18%-36%. Figure 6 ).and Compared to OnePot Pro DNAFragmentation Reagent (#12619), the embodiments of this invention show a significant improvement in enzyme cleavage site bias. Examples 1-6, using different enzyme compositions, resulted in reduced base fluctuations at the cleavage sites and decreased bias. Furthermore, Examples 4, 5, and 6, with the addition of disaccharides or disaccharide derivatives, showed even better results than the simple enzyme compositions, further reducing base fluctuations at the cleavage sites and decreasing bias. The enzyme composition and reaction solution used in Example 6 were the optimal combination, resulting in minimal base fluctuations at the cleavage sites and the lowest bias.

[0124] The closer the coverage of different GC regions is to the average value of 1, the smaller the GC bias and the better the uniformity. The GC bias results of Examples 1-6 are as follows. Figure 8-13 As shown.

[0125] Results: GC preference results for Examples 1-6 ( Figures 8-13 Both are better than Yisheng Bio. OnePotPro DNAFragmentation Reagent(#12619)( Figure 14 This demonstrates that the enzyme composition and reaction solution of the present invention can ultimately obtain DNA fragments with low bias, resulting in libraries with higher uniformity and broad application prospects in next-generation sequencing. Specifically, the GC bias in Example 4 is better than that in Example 1, the GC bias in Example 5 is better than that in Example 2, and the GC bias in Example 6 is better than that in Example 3. Example 6 exhibits the lowest GC bias, resulting in the best outcome, indicating that the enzyme composition and reaction solution of Example 6 can ultimately obtain DNA fragments with low bias, resulting in libraries with the best uniformity and broad application prospects in next-generation sequencing.

[0126] In summary, in the next-generation sequencing (NGS) library construction process, the first steps—fragmentation, end repair, and A-tailing—play a crucial role in the final library quality. This invention explores the enzyme composition and corresponding reaction solution for these steps, synergistically optimizing the concentration, type, and quantity of each component. This ultimately solves the problem of poor library uniformity produced by enzyme digestion under high input levels, significantly reducing restriction enzyme site bias and GC bias. Furthermore, by conducting a complete NGS sequencing experiment on the DNA samples to be tested, the effectiveness of the enzyme composition and reaction solution provided by this invention is verified from the final sequencing results, further demonstrating its practical application value in NGS production.

[0127] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a sequencing library, characterized in that, The method includes: adding the target nucleic acid to a mixture of an enzyme composition and an enzyme composition reaction solution, incubating, and performing fragmentation, end repair, 5' phosphorylation, and 3' A-tailing of the target nucleic acid; The enzyme composition includes DNase I, endonuclease IV, Taq DNA polymerase, the Klenow fragment of DNA polymerase I, T4 DNA polymerase, and T4 polynucleotide kinase; The enzyme composition reaction solution includes sucrose and / or stachyose.

2. The method according to claim 1, characterized in that, The concentration of DNase I in the enzyme composition is 0.1-3 U / μL.

3. The method according to claim 1, characterized in that, The concentration of endonuclease IV in the enzyme composition is 0.005-2 U / μL.

4. The method according to claim 1, characterized in that, The concentration of Taq DNA polymerase in the enzyme composition is 2-35 U / μL.

5. The method according to claim 1, characterized in that, The concentration of the Klenow fragment of DNA polymerase I in the enzyme composition is 0.01-7.8 U / μL.

6. The method according to claim 1, characterized in that, The concentration of T4 DNA polymerase in the enzyme composition is 0.3-40 U / μL.

7. The method according to claim 1, characterized in that, The concentration of T4 polynucleotide kinase in the enzyme composition is 0.1-3 U / μL.

8. The method according to claim 1, characterized in that, The concentration of sucrose in the reaction solution is 50-120 mM.

9. The method according to claim 1, characterized in that, The concentration of stachyose in the reaction solution is 30-80 mM.

10. The method according to claim 1, characterized in that, The reaction solution also includes buffer solution, metal salt ions, detergent, ATP, dATP and dNTP.

11. The method according to claim 10, characterized in that, The buffer solution comprises any one or a combination of at least two of the following: tris(hydroxymethyl)aminomethane acetate, tris(hydroxymethyl)aminomethane hydrochloride, or 4-hydroxyethylpiperazine ethanesulfonic acid.

12. The method according to claim 11, characterized in that, The reaction solution contains 50-200 mM of tris(hydroxymethyl)aminomethane acetate and has a pH of 7-8.

5.

13. The method according to claim 11, characterized in that, The reaction solution contains 100-150 mM of trihydroxymethylaminomethane hydrochloride and has a pH of 7-9.

14. The method according to claim 11, characterized in that, The reaction solution contains 100-180 mM of 4-hydroxyethylpiperazine ethanesulfonic acid and has a pH of 7-9.

15. The method according to claim 10, characterized in that, The metal salt ions include any one or a combination of at least two of CaCl2, MgCl2, ZnCl2, NaCl, or KCl.

16. The method according to claim 15, characterized in that, The CaCl2 content in the reaction solution is 15-50 mM.

17. The method according to claim 15, characterized in that, The MgCl2 content in the reaction solution is 35-200 mM.

18. The method according to claim 15, characterized in that, The ZnCl2 content in the reaction solution is 20-80 mM.

19. The method according to claim 15, characterized in that, The NaCl content in the reaction solution is 20-200 mM.

20. The method according to claim 15, characterized in that, The KCl content in the reaction solution is 30-80 mM.

21. The method according to claim 10, characterized in that, The detergent includes any one or a combination of at least two of DTT, Triton X-100, Tween-20, or NP-40.

22. The method according to claim 21, characterized in that, The DTT content in the reaction solution is 10-50 mM.

23. The method according to claim 21, characterized in that, The content of Triton X-100 in the reaction solution is 0.1-1%.

24. The method according to claim 21, characterized in that, The content of Tween-20 in the reaction solution is 0.01-1%.

25. The method according to claim 21, characterized in that, The content of NP-40 in the reaction solution is 0.01-1%.

26. The method according to claim 10, characterized in that, The dATP content in the reaction solution is 0.2-8 mM.

27. The method according to claim 26, characterized in that, The dATP content in the reaction solution is 2-4 mM.

28. The method according to claim 10, characterized in that, The dNTP content in the reaction solution is 0.02-2 mM.

29. The method according to claim 28, characterized in that, The dNTP content in the reaction solution is 0.02-1 mM.

30. The method according to claim 10, characterized in that, The ATP content in the reaction solution is 5-15 mM.

31. A kit for constructing sequencing libraries, characterized in that, The kit comprises the enzyme composition and reaction solution of claim 1, wherein the enzyme composition comprises DNase I, endonuclease IV, Taq DNA polymerase, Klenow fragment of DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase; The enzyme composition reaction solution includes sucrose and / or stachyose.

32. The reagent kit according to claim 31, characterized in that, The kit also includes sequencing adapters and ligation reaction reagents.

33. The reagent kit according to claim 31, characterized in that, The concentration of DNase I in the enzyme composition is 0.1-3 U / μL.

34. The reagent kit according to claim 31, characterized in that, The concentration of endonuclease IV in the enzyme composition is 0.005-2 U / μL.

35. The reagent kit according to claim 31, characterized in that, The concentration of Taq DNA polymerase in the enzyme composition is 2-35 U / μL.

36. The reagent kit according to claim 31, characterized in that, The concentration of the Klenow fragment of DNA polymerase I in the enzyme composition is 0.01-7.8 U / μL.

37. The reagent kit according to claim 31, characterized in that, The concentration of T4 DNA polymerase in the enzyme composition is 0.3-40 U / μL.

38. The reagent kit according to claim 31, characterized in that, The concentration of T4 polynucleotide kinase in the enzyme composition is 0.1-3 U / μL.

39. The reagent kit according to claim 31, characterized in that, The concentration of sucrose in the reaction solution is 50-120 mM.

40. The kit according to claim 31, characterized in that, The concentration of stachyose in the reaction solution is 30-80 mM.

41. The reagent kit according to claim 31, characterized in that, The reaction solution also includes buffer solution, metal salt ions, detergent, ATP, dATP and dNTP.

42. The kit according to claim 41, characterized in that, The buffer solution comprises any one or a combination of at least two of the following: tris(hydroxymethyl)aminomethane acetate, tris(hydroxymethyl)aminomethane hydrochloride, or 4-hydroxyethylpiperazine ethanesulfonic acid.

43. The reagent kit according to claim 42, characterized in that, The reaction solution contains 50-200 mM of tris(hydroxymethyl)aminomethane acetate and has a pH of 7-8.

5.

44. The kit according to claim 42, characterized in that, The reaction solution contains 100-150 mM of trihydroxymethylaminomethane hydrochloride and has a pH of 7-9.

45. The reagent kit according to claim 42, characterized in that, The reaction solution contains 100-180 mM of 4-hydroxyethylpiperazine ethanesulfonic acid and has a pH of 7-9.

46. ​​The kit according to claim 41, characterized in that, The metal salt ions include any one or a combination of at least two of CaCl2, MgCl2, ZnCl2, NaCl, or KCl.

47. The kit according to claim 46, characterized in that, The CaCl2 content in the reaction solution is 15-50 mM.

48. The reagent kit according to claim 46, characterized in that, The MgCl2 content in the reaction solution is 35-200 mM.

49. The reagent kit according to claim 46, characterized in that, The ZnCl2 content in the reaction solution is 20-80 mM.

50. The kit according to claim 46, characterized in that, The NaCl content in the reaction solution is 20-200 mM.

51. The reagent kit according to claim 46, characterized in that, The KCl content in the reaction solution is 30-80 mM.

52. The kit according to claim 41, characterized in that, The detergent includes any one or a combination of at least two of DTT, Triton X-100, Tween-20, or NP-40.

53. The reagent kit according to claim 52, characterized in that, The DTT content in the reaction solution is 10-50 mM.

54. The reagent kit according to claim 52, characterized in that, The content of Triton X-100 in the reaction solution is 0.1-1%.

55. The reagent kit according to claim 52, characterized in that, The content of Tween-20 in the reaction solution is 0.01-1%.

56. The reagent kit according to claim 52, characterized in that, The content of NP-40 in the reaction solution is 0.01-1%.

57. The reagent kit according to claim 41, characterized in that, The dATP content in the reaction solution is 0.2-8 mM.

58. The reagent kit according to claim 57, characterized in that, The dATP content in the reaction solution is 2-4 mM.

59. The reagent kit according to claim 41, characterized in that, The dNTP content in the reaction solution is 0.02-2 mM.

60. The reagent kit according to claim 59, characterized in that, The dNTP content in the reaction solution is 0.02-1 mM.

61. The reagent kit according to claim 41, characterized in that, The ATP content in the reaction solution is 5-15 mM.

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