CN116286726A - A low-preference DNA fragmentation enzyme composition and its reaction solution - Google Patents

Abstract

The invention discloses an enzyme composition for low-preference DNA fragmentation and a reaction solution thereof. The enzyme composition comprises endonuclease, nucleotide kinase and DNA polymerase, wherein the endonuclease comprises any one or a combination of at least two of DNase I, thermostable high salt endonuclease HL-SAN or endonuclease 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, klenow fragment of DNA polymerase I or T4DNA polymerase. The invention develops a module suitable for genome fragmentation, end repair and A tail addition by a one-tube method, comprises an enzyme composition and a reaction solution thereof, aims at the problems and the defects existing in the current enzyme method library establishment, and aims at the problems and the defects of the enzyme method library establishment, and the problems are solved by optimizing and exploring each component and matching with a proper connecting module.

Description

A low-preference DNA fragmentation enzyme composition and its reaction solution

technical field

The invention belongs to the field of biotechnology, and relates to an enzyme composition for DNA fragmentation with low preference and a reaction liquid thereof.

Background technique

In recent decades, DNA sequencing technology has been applied to molecular biology-related research, which has greatly promoted the rapid development of this field. Although the first-generation DNA sequencing technology has a read length of up to 1000bp and an accuracy of 99.99% to help researchers complete a large amount of sequencing work, the shortcomings of the first-generation sequencing technology, such as slow speed, high cost and low throughput, are difficult to support the growth of sequencing demand. Next-generation sequencing (NGS), also known as high-throughput sequencing (HTS), has been widely used in scientific research and clinical practice because of its low cost and high throughput. and other fields.

The main process of second-generation sequencing is divided into three parts, including library construction, gene sequencing and bioinformatics analysis. Among them, library construction is the initial step of NGS sequencing, and the generated library has an important impact on the final sequencing results. Therefore, it is particularly important to do a good job in library construction to improve the quality of the library. Based on the short read length characteristics of next-generation sequencing, the first step in DNA library construction needs to fragment the metagenomic group. Currently, there are mechanical methods and fragmentation enzyme methods that can complete the fragmentation of genomic DNA. Compared with the high requirements for equipment and long operating time of the traditional mechanical method, fragmentation enzymatic method has been widely used due to its advantages of simple operation, short time and low cost.

However, compared with the unbiased random cutting of the traditional mechanical fragmentation method, the fragmentation enzyme fragmentation method will preferentially select the enzyme cutting site on the DNA double strand for cutting, which leads to the abundance of random fragments in the library. The degree and diversity of the library are reduced, and the uniformity of the output library is poor, which will cause large deviations in the sequencing depth of different regions of the gene, so that some gene loci with low abundance are difficult to detect during sequencing, and may occur The case of missed detection. Using the method of increasing the sequencing depth can reduce the missed detection sites, but it will cause a lot of data waste.

In summary, the current fragmentation enzyme fragmentation method has low abundance and diversity of random fragments in the library, poor uniformity of the output library, and large deviations in the sequencing depth of different regions of the gene. Gene loci with low abundance are difficult to detect, and there are problems such as missed detection. Provides an enzyme composition for DNA fragmentation and its reaction solution, suitable for one-tube genome fragmentation, end repair and A-tailing modules, to obtain a library with low bias and higher output uniformity, and has been It has become one of the urgent problems to be solved in the field of biotechnology.

Contents of the invention

Aiming at the deficiencies and actual needs of the prior art, the present invention provides a low-preference DNA fragmentation enzyme composition and its reaction solution, which solves the problem of the abundance and diversity of random fragments in the library existing in the current fragmentation enzyme fragmentation method. The uniformity of the library is low, the uniformity of the output library is poor, there is a large deviation in the sequencing depth of different regions of the gene, and some gene loci with low abundance are difficult to detect during sequencing, and there are problems such as missed detection. The enzyme composition and its reaction solution provided by the invention have good versatility and are suitable for various types of next-generation sequencing reactions.

To achieve this purpose of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides an enzyme composition for DNA fragmentation with low preference, the enzyme composition includes endonuclease, nucleotide kinase and DNA polymerase, and the endonuclease includes DNaseI, Any one or a combination of at least two of thermolabile high-salt-resistant endonuclease HL-SAN or endonuclease IV, said nucleotide kinase comprising T4 polynucleotide kinase, said DNA The polymerase includes any one or a combination of at least two of Taq DNA polymerase, Klenow fragment of DNA polymerase I or T4 DNA polymerase.

The invention develops a module suitable for one-tube genome fragmentation, end repair, 5' phosphorylation and 3' adding A tail, including an enzyme composition and its reaction solution, which can reduce the preference of DNA fragmentation, and is aimed at the current enzymatic method Problems and deficiencies in library construction, through optimization and exploration of each component, with a suitable connection module, the final product obtained has low preference, and a library with higher output uniformity, which has a wide range of applications in next-generation sequencing prospect.

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

(1) Compositions of DNase I, Taq DNA polymerase, Klenow fragment of DNA polymerase I and T4 polynucleotide kinase;

(2) Compositions of heat-labile high-salt-resistant endonuclease HL-SAN, Taq DNA polymerase, Klenow fragment of DNA polymerase I and T4 polynucleotide kinase;

(3) Composition of DNase I, endonuclease IV, Taq DNA polymerase, Klenow fragment of DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase.

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

The specific point values in the above 0.1-3 can be selected from 0.1, 0.5, 1, 1.5, 2, 2.5, 3 and so on.

Preferably, the concentration of the heat-labile high-salt-tolerant endonuclease HL-SAN in the enzyme composition is 0.01-3 U/μL.

The specific point values in the above 0.01-3 can be selected as 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.

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

The specific point value in the above 0.005-2 can be selected as 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.

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

The specific point values in the above 2-35 can be selected from 2, 6, 9, 12, 20, 25, 29, 30, 31, 32, 33, 34, 35, etc.

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

The specific point value in the above 0.01-7.8 can be selected as 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 and so on.

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

The specific point value in the above-mentioned 0.3-40 can be selected as 0.3, 0.5, 1, 2, 3, 8, 10, 20, 30, 32, 33, 34, 36, 38, 39, 40, etc.

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

The specific point values in the above 0.1-3 can be selected as 0.2, 0.5, 1, 1.5, 2.5, etc.

In a second aspect, the present invention provides a reaction solution of an enzyme composition, the reaction solution comprising: disaccharides and/or disaccharide derivatives.

The disaccharide or disaccharide derivatives in the reaction liquid of the present invention can maintain the structural stability of the protein, enhance the ability of the protein functional domain of the enzyme to non-specifically recognize and cut the site on the DNA double strand, and can promote the synergy between enzyme compositions effect, further reducing the bias of library generation.

Preferably, the disaccharide comprises sucrose.

Preferably, the disaccharide derivatives include stachyose.

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

You can choose 50, 60, 70, 80, 90, 100, 110, 120, etc. for the specific point value in the above-mentioned 50-120.

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

The specific point value in the above-mentioned 30-80 can choose 30, 40, 50, 60, 70, 80, etc.

Preferably, the reaction solution further includes buffer, metal salt ions, detergent, ATP, dATP and dNTP.

Preferably, the buffer solution includes any one or a combination of at least two of tris(hydroxymethylaminomethane) acetate, tris(hydroxymethylaminomethane) hydrochloride or 4-hydroxyethylpiperazineethanesulfonic acid.

Preferably, the content of Tris in the reaction solution is 50-200mM, and the pH is 7-8.5.

50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 190, 200, etc. can be selected for the specific point value in the above-mentioned 50-200.

Preferably, the content of tris hydrochloride in the reaction solution is 100-150 mM, and the pH is 7-9.

You can choose 7, 8, 9, etc. for the specific point value in the above 7-9.

You can choose 100, 110, 120, 130, 140, 150, etc. for the specific point value in the above-mentioned 100-150.

Preferably, preferably, the content of 4-hydroxyethylpiperazineethanesulfonic acid in the reaction solution is 100-180 mM, and the pH is 7-9.

You can choose 7, 8, 9, etc. for the specific point value in the above 7-9.

The specific point value in the above 100-180 can be selected as 100, 110, 120, 130, 140, 150, 160, 180 and so on.

Preferably, the metal salt ions include any one or a combination of at least two of CaCl ₂ , MgCl ₂ , ZnCl ₂ , NaCl or KCl.

Preferably, the content of _CaCl2 in the reaction solution is 15-50mM.

The specific point value in the above 20-50 can be selected as 15, 30, 40, 50 and so on.

Preferably, the content of MgCl ₂ in the reaction solution is 35-200mM.

The specific point values in the above 50-200 can be selected from 35, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 190, 200 and so on.

Preferably, the content of ZnCl ₂ in the reaction solution is 20-80mM.

The specific point value in the above-mentioned 20-80 can choose 20, 30, 40, 50, 60, 70, 80 and so on.

Preferably, the content of NaCl in the reaction solution is 20-200mM.

The specific point value in the above-mentioned 20-200 can choose 20, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 190, 200 and so on.

Preferably, the content of KCl in the reaction solution is 30-80mM.

The specific point value in the above-mentioned 30-80 can choose 30, 40, 50, 60, 70, 80, etc.

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

Preferably, the content of DTT in the reaction solution is 10-50mM.

You can choose 10, 20, 30, 40, 50, etc. for the specific point value in the above-mentioned 10-50.

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

The specific point value in the above 0.1-1 can be selected as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 and so on.

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

The specific point value in the above 0.01-1 can be selected as 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 and so on.

Preferably, the content of NP-40 in the reaction solution is 0.01-1%.

The specific point value in the above 0.01-1 can be selected as 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 and so on.

Preferably, the content of dATP in the reaction solution is 0.2-8mM, preferably 2-4mM.

The specific point values in the above 0.2-8 can be selected from 0.2, 0.3, 0.4, 1, 2, 3, 4, 5, 6, 7, 8, etc.

The specific point values in the above 2-4 can be selected as 2, 3, 4, etc.

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

The specific point values in the above 0.02-2 can be selected as 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.

The content of ATP in the reaction solution is 5-15mM.

The specific point values in the above 5-10 can be selected as 5, 6, 7, 8, 9, 10, 11, 15 and so on.

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

In a fourth aspect, the present invention provides a kit for constructing a sequencing library, the kit including the enzyme composition described in the first aspect or the reaction solution described in the second aspect.

Preferably, the kit also includes sequencing adapters and ligation reagents.

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

(1) The one-tube genome fragmentation, end repair and A-tailing modules developed by the present invention, including the enzyme composition and its reaction solution, aim at the problems and deficiencies of the current enzymatic library construction, by optimizing each component Exploration, used with the connection module, and through the completion of the whole process of next-generation sequencing experiments on the DNA samples to be tested, the effects of the enzyme composition and its reaction solution provided by the present invention are verified from the final sequencing results, further confirming its role in next-generation sequencing It has practical application value in production.

(2) The scheme provided by the present invention has good versatility and is applicable to various types of next-generation sequencing reactions. In the application of PCRfree library construction, the target gene undergoes two steps of fragmentation, end repair, addition of A and linker ligation to obtain a non-PCR-amplified library. The advantage of constructing a PCR free library for sequencing is that it is faster and more efficient, and can avoid problems such as poor fidelity caused by PCR amplification. The enzyme composition and its reaction solution provided by the present invention solve the problem of High input restriction site preference and high GC preference.

Description of drawings

Fig. 1 is the base distribution figure of embodiment 1 restriction enzyme digestion library construction sequencing reads;

Fig. 2 is the base distribution figure of embodiment 2 enzyme digestion library construction sequencing reads;

Fig. 3 is the base distribution figure of embodiment 3 enzyme digestion library construction sequencing reads;

Fig. 4 is the base distribution figure of embodiment 4 restriction endonuclease library construction sequencing reads;

Fig. 5 is the base distribution figure of embodiment 5 enzyme digestion library construction sequencing reads;

Fig. 6 is the base distribution figure of embodiment 6 enzyme digestion library construction sequencing reads;

Figure 7 is the base distribution map of 12619 enzyme digestion library construction and sequencing reads;

Fig. 8 is the coverage distribution diagram of different GC regions for enzyme digestion library construction and sequencing in Example 1;

Figure 9 is a distribution map of the coverage of different GC regions for enzyme digestion library construction and sequencing in Example 2;

Fig. 10 is the coverage distribution diagram of different GC regions for enzyme digestion library construction and sequencing in Example 3;

Figure 11 is a distribution map of the coverage of different GC regions for enzyme digestion library construction and sequencing in Example 4;

Figure 12 is a distribution map of the coverage of different GC regions for enzyme digestion library construction and sequencing in Example 5;

Figure 13 is a distribution map of the coverage of different GC regions for enzyme digestion library construction and sequencing in Example 6;

Figure 14 is the coverage distribution map of different GC regions for 12619 enzyme digestion library construction and sequencing.

Detailed ways

In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through formal channels.

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

Table 1

Example 1

This example provides an enzyme composition and its reaction solution: DNase I 1.5 U/μL, Taq DNA polymerase 1 U/μL, DNA polymerase I Klenow fragment 2 U/μL, T4 polynucleotide kinase 1 U/μL, Reaction solution: Tris-Acetate 75mM pH7.5, CaCl ₂ 20 mM, MgCl ₂ 45 mM, NaCl 20 mM, ZnCl ₂ 50 mM, Triton X-1000.1%, Tween-200.01%, dATP 2mM, dNTP 0.2mM, ATP 9mM.

Example 2

This example provides an enzyme composition and its reaction solution: heat-labile high-salt-tolerant endonuclease HL-SAN 0.5U/μL, Taq DNA polymerase 5U/μL, DNA polymerase I Klenow fragment 2U/μL μL, T4 polynucleotide kinase 1U/μL; reaction solution: HEPES 120mM pH7.5, CaCl ₂ 35 mM, MgCl ₂ 35mM, NaCl 30mM, KCl 60mM, TritonⅩ-1000.1%, Tween-200.05%, NP-400.05 %, dATP 2mM, dNTP 0.2mM, ATP 15mM.

Example 3

This embodiment provides an enzyme composition and its reaction solution: DNase I 1U/μL, endonuclease IV 0.05U/μL, Taq DNA polymerase 9U/μL, DNA polymerase I Klenow fragment 2U/μL, T4 DNA polymerase Enzyme 0.5U/μL, T4 polynucleotide kinase 2U/μL; reaction solution: Tris-HCl 100mM pH7.5, CaCl ₂ 15 mM, MgCl ₂ 50 mM, NaCl 30mM, NP-400.02%, Tween-200.01% , dATP 2mM, dNTP 0.2mM, ATP 9mM.

Example 4

This example provides an enzyme composition and its reaction solution: DNase I 1.5 U/μL, Taq DNA polymerase 1 U/μL, DNA polymerase I Klenow fragment 2 U/μL, T4 polynucleotide kinase 1 U/μL, Reaction solution: sucrose 50mM, Tris-Acetate 75mM pH7.5, CaCl ₂ 20 mM, MgCl ₂ 45 mM, NaCl 20mM, ZnCl ₂ 50 mM, TritonⅩ-1000.1%, Tween-200.01%, dATP 2mM, dNTP 0.2mM, ATP 9mM. The difference between this example and Example 1 is that 50 mM sucrose is added to the reaction solution.

Example 5

This example provides an enzyme composition and its reaction solution: heat-labile hypersalt-resistant endonuclease HL-SAN 0.5U/μL, Taq DNA polymerase 5U/μL, DNA polymerase I Klenow fragment 2U/μL μL, T4 polynucleotide kinase 2U/μL; reaction solution: sucrose 120mM, HEPES 120mM pH7.5, CaCl ₂ 35 mM, MgCl ₂ 35mM, NaCl 30mM, KCl 60mM, TritonⅩ-1000.1%, Tween-200.05%, NP-400.05%, dATP 2mM, dNTP 0.2mM, ATP 15mM. The difference between this example and Example 2 is that 120 mM sucrose is added to the reaction solution.

Example 6

DNase Ⅰ 1U/μL, endonuclease Ⅳ 0.05U/μL, Taq DNA polymerase 9U/μL, DNA polymerase Ⅰ Klenow fragment 2U/μL, T4 DNA polymerase 0.5U/μL, T4 polynucleotide kinase 12U /μL; Reaction solution: stachyose 30mM, Tris-HCl 100mM pH7.5, CaCl ₂ 15 mM, MgCl ₂ 50mM, NaCl 30mM, NP-400.02%, Tween-200.01%, dATP 2mM, dNTP 0.2mM, ATP 9mM . The only difference between this example and Example 3 is that 30 mM stachyose is added to the reaction solution.

Example 7

Construct PCR free sequencing library.

Rapid DNA Ligation Module for kit(#13580)和华大智造MGIEasyPFAdapters(#1000013460)进行接头连接反应，使用/>

Smarter DNAClean Beads(#12600)对接头连接产物进行纯化分选，获得可供测序的PCR free文库。In this example, human female gDNA is used as a template, and the initial input amount is 1000ng. Different enzyme compositions and matching reaction solutions in Examples 1-6 are used to fragment/end repair/5' phosphorylation/3 'Add A tail, with Yisheng creatures

Rapid DNA Ligation Module for kit (#13580) and MGI MGIEasyPFAdapters (#1000013460) for adapter ligation reaction, use />

Smarter DNAClean Beads (#12600) were used to purify and sort adapter ligation products to obtain a PCR free library for sequencing.

OnePot Pro DNA FragmentationReagent(#12619)进行对比。对gDNA模板进行片段化/末端修复/5’磷酸化/3’加A尾，搭配翌圣生物/>

Rapid DNA Ligation Module for kit(#13580)和华大智造MGIEasyPF Adapters(#1000013460)进行接头连接反应，使用翌圣生物/>

Smarter DNAClean Beads(#12600)对接头连接产物进行纯化分选，构建PCR free文库，具体操作步骤参考说明书。This example also uses the commercial Yisheng biological

OnePot Pro DNA FragmentationReagent (#12619) for comparison. Fragmentation/end repair/5'phosphorylation/3'adding A tail on gDNA template, with Yisheng Bio>

Rapid DNA Ligation Module for kit (#13580) and MGI MGIEasyPF Adapters (#1000013460) for linker ligation reaction, using Yisheng Biology/>

Smarter DNAClean Beads (#12600) was used to purify and sort adapter ligation products to construct a PCR free library. Please refer to the manual for specific operation steps.

The target DNA fragmentation/end repair/5' phosphorylation/3' plus A tail reaction system is shown in Table 2, wherein the formulations of the enzyme composition are respectively used in Examples 1-6, and the reaction conditions are incubation at 30°C for 15 minutes, Incubate at 65°C for 20min.

Table 2

components Dosage target DNA 1000ng enzyme composition 5μL The reaction solution 10 μL 1×TE Make up to 60μL

The resulting fragmentation/end repair/5' phosphorylation/3' plus A tail product was directly subjected to the adapter ligation reaction, the reaction system was shown in Table 3, and the reaction conditions were incubation at 20°C for 15 minutes.

table 3

components Dosage Fragmentation/end repair/5' phosphorylation/3' A-tailing 60μL Ligation Buffer (Ligation Enhancer) 30μL MGIEasy PF Adapters 5μL Ligase (Rapid T4 DNA Ligase2.0) 5μL

Smarter DNA Clean Beads进行分选，分选比例为0.7/0.2×，22μL无菌ddH₂O洗脱，回收20μL，即为可供上机测序的PCRfree文库，测序使用MGI DNBSEQ-T7平台，模式为PE150。The adapter ligation products obtained after incubation were used

Smarter DNA Clean Beads were sorted, the sorting ratio was 0.7/0.2×, 22 μL sterile ddH ₂ O was eluted, and 20 μL was recovered, which was the PCRfree library for sequencing on the machine. The sequencing used the MGI DNBSEQ-T7 platform, and the mode was PE150.

Analyze the sequencing data, use fastp 0.23.1 to filter the original data containing adapter sequences and low-quality Reads, and obtain high-quality sequencing data (Clean Reads), each sample takes the same amount of Clean Reads data, using FastQC 0.11.9 After analyzing the selected Clean Reads, the size of base fluctuations corresponding to the Perbase sequencecontent index in the analysis results is used as an index of restriction restriction preference. The greater the fluctuation degree, the more serious restriction restriction is. The experimental analysis results are shown in Figure 1-7.

Use bwa0.7.17 to compare the selected Clean Reads with the hg38 reference genome, use Samtools1.16.1 to compare and sort the files, use the Collect GC Bias Metrics module of picard.jar 1.86.0 to analyze the coverage information of different GC regions of the genome, According to the displayed results of the coverage of different GC regions of the genome, the fragmentation preference of enzyme digestion for regions with different GC content of the genome can be judged. The experimental analysis results are shown in Figure 8-14.

OnePot Pro DNA FragmentationReagent(#12619)酶切位点碱基波动值为4-50％；本发明中应用实施例1酶切位点碱基波动值为12-42％(图1)，对应添加二糖的实施例4酶切位点碱基波动值为16％-38％(图4)；实施例2酶切位点碱基波动值为16％-40％(图2)，对应添加二糖的实施例5酶切位点碱基波动值为18％-39％(图5)；实施例3酶切位点碱基波动值为15％-40％(图3)，对应添加二糖衍生物的实施例6酶切位点碱基波动值为18％-36％(图6)。和/>

OnePot Pro DNAFragmentation Reagent(#12619)相比，本发明中的实施例在酶切位点偏好性上有很大程度的改善。其中实施例1-6选用不同的酶组合物结果酶切位点碱基波动减小，偏好性降低；进一步地，实施例4、实施例5、实施例6分别添加二糖或二糖衍生物结果比单纯酶组合物效果更好，酶切位点碱基波动进一步减小，偏好性进一步降低，实施例6选用的酶组合物及其反应液为最优选的组合，结果酶切位点碱基波动最小，偏好性最低。Result: From Figure 7, it can be seen that Yisheng biological

OnePot Pro DNA FragmentationReagent (#12619) has a base fluctuation value of 4-50% at the enzyme cutting site; in the present invention, the base fluctuation value of the enzyme cutting site in Example 1 is 12-42% (Figure 1), corresponding to adding two The base fluctuation value of the enzyme cutting site in Example 4 of the sugar is 16%-38% (Figure 4); the base fluctuation value of the enzyme cutting site in Example 2 is 16%-40% (Figure 2), corresponding to the addition of disaccharides The base fluctuation value of the enzyme cutting site in Example 5 is 18%-39% (Figure 5); the base fluctuation value of the enzyme cutting site in Example 3 is 15%-40% (Figure 3), corresponding to the addition of disaccharide derivatives The base fluctuation value of the enzyme cleavage site in Example 6 of the product is 18%-36% (FIG. 6). and />

Compared with OnePot Pro DNA Fragmentation Reagent (#12619), the embodiments of the present invention have greatly improved the restriction site preference. Among them, the selection of different enzyme compositions in Examples 1-6 results in reduced base fluctuations and reduced preferences for enzyme cleavage sites; further, in Example 4, Example 5, and Example 6, disaccharides or disaccharide derivatives are added respectively As a result, the effect is better than that of the simple enzyme composition, the base fluctuation of the restriction site is further reduced, and the preference is further reduced. The enzyme composition and its reaction solution selected in Example 6 are the most preferred combination. The base fluctuation is the smallest, and the preference is the lowest.

The closer the coverage of different GC regions is to the average value 1, the smaller the GC bias and the better the uniformity. The GC bias results of Examples 1-6 are shown in Figures 8-13.

OnePot Pro DNAFragmentation Reagent(#12619)(图14)说明利用本发明的酶组合物及反应液能够最终获得具有低偏好性的DNA片段，产出的文库均一性更高，在二代测序中具有广阔的应用前景。其中实施例4的GC偏好性相较于实施例1结果更好，实施例5的GC偏好性相较于实施例2结果更好，实施例6的GC偏好性相较于实施例3结果更好。实施例6的GC偏好性最低，结果最好，说明实施例6的酶组合物及反应液能够最终获得具有低偏好性的DNA片段，产出的文库均一性最佳，在二代测序中具有广阔的应用前景。Results: The GC preference results of Examples 1-6 (Figure 8-Figure 13) are better than those of Yisheng Biology

OnePot Pro DNA Fragmentation Reagent (#12619) (Figure 14) shows that the use of the enzyme composition and reaction solution of the present invention can finally obtain DNA fragments with low preference, and the output library has higher uniformity, which has broad application potential in next-generation sequencing. application prospects. The GC preference of Example 4 is better than that of Example 1, the GC preference of Example 5 is better than that of Example 2, and the GC preference of Example 6 is better than that of Example 3. good. Example 6 has the lowest GC preference and the best result, indicating that the enzyme composition and reaction solution of Example 6 can finally obtain DNA fragments with low preference, and the output library has the best uniformity, and has the advantages in next-generation sequencing. Broad application prospects.

To sum up, in the process of next-generation sequencing library construction, the first step of fragmentation, end repair, and A tailing reaction plays a very important role in the quality of the final library. The present invention adopts the enzyme composition for this step and its corresponding reaction solution, and the synergistic optimization of the concentration, type, and quantity of each component finally solved the problem of poor uniformity of the library produced by the enzyme digestion method under high input, and greatly reduced the Library restriction site preference and GC preference. And by completing the whole process experiment of next-generation sequencing on the DNA sample to be tested, the effects of the enzyme composition and its reaction solution provided by the present invention are verified from the final sequencing results, which further proves that it has practical application value in the production of next-generation sequencing .

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

Claims (10)

1. an enzyme composition of low preference DNA fragmentation, is characterized in that, described enzyme composition comprises endonuclease, nucleotide kinase and DNA polymerase; The endonuclease includes any one or a combination of at least two of DNase I, heat-labile high-salt-resistant endonuclease HL-SAN or endonuclease IV; The nucleotide kinases include T4 polynucleotide kinases; The DNA polymerase includes any one or a combination of at least two of Taq DNA polymerase, Klenow fragment of DNA polymerase I or T4 DNA polymerase. 2. enzyme composition according to claim 1, is characterized in that, described enzyme composition comprises any one of following composition: (1) Compositions of DNase I, Taq DNA polymerase, Klenow fragment of DNA polymerase I and T4 polynucleotide kinase; (2) Compositions of thermolabile high-salt-resistant endonuclease HL-SAN, TaqDNA polymerase, Klenow fragment of DNA polymerase I and T4 polynucleotide kinase; (3) Composition of DNase I, endonuclease IV, Taq DNA polymerase, Klenow fragment of DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase. 3. The enzyme composition according to claim 1 or 2, characterized in that, the concentration of DNase I in the enzyme composition is 0.1-3U/μL; Preferably, the concentration of the heat-labile high-salt-resistant endonuclease HL-SAN in the enzyme composition is 0.01-3U/μL; Preferably, the concentration of endonuclease IV in the enzyme composition is 0.005-2U/μL; Preferably, the concentration of TaqDNA polymerase in the enzyme composition is 2-35U/μL; Preferably, the concentration of the Klenow fragment of DNA polymerase I in the enzyme composition is 0.01-7.8 U/μL; Preferably, the concentration of T4 DNA polymerase in the enzyme composition is 0.3-40U/μL; Preferably, the concentration of T4 polynucleotide kinase in the enzyme composition is 0.1-3 U/μL. 4. A reaction solution of an enzyme composition, characterized in that the reaction solution comprises: disaccharides and/or disaccharide derivatives; Preferably, the disaccharide comprises sucrose; Preferably, the disaccharide derivatives include stachyose; Preferably, the concentration of sucrose in the reaction solution is 50-120mM; Preferably, the concentration of stachyose in the reaction solution is 30-80mM. 5. The reaction solution according to claim 4, characterized in that, the reaction solution further comprises buffer, metal salt ions, detergent, ATP, dATP and dNTP. 6. reaction solution according to claim 5, is characterized in that, described damping fluid comprises Tris hydroxymethyl amino methane acetate, tris hydroxymethyl amino methane hydrochloride or 4-hydroxyethylpiperazine ethyl sulfonate Any one or a combination of at least two of the acids; Preferably, the content of Tris in the reaction solution is 50-200mM, and the pH is 7-8.5; Preferably, the content of Tris hydrochloride in the reaction solution is 100-150mM, and the pH is 7-9; Preferably, the content of 4-hydroxyethylpiperazineethanesulfonic acid in the reaction solution is 100-180 mM, and the pH is 7-9. 7. The reaction solution according to claim 5 or 6, wherein the metal salt ions include any one or a combination of at least two of CaCl ₂ , MgCl ₂ , ZnCl ₂ , NaCl or KCl; Preferably, the content of _CaCl in the reaction solution is 15-50mM; Preferably, the content of _MgCl in the reaction solution is 35-200mM; Preferably, the _ZnCl content in the reaction solution is 20-80mM; Preferably, the content of NaCl in the reaction solution is 20-200mM; Preferably, the content of KCl in the reaction solution is 30-80mM. 8. The reaction solution according to any one of claims 5-7, characterized in that, the detergent comprises any one of DTT, TritonX-100, Tween-20 or NP-40 or at least a combination of the two; Preferably, the content of DTT in the reaction solution is 10-50mM; Preferably, the content of TritonX-100 in the reaction solution is 0.1-1%; Preferably, the content of Tween-20 in the reaction solution is 0.01-1%; Preferably, the content of NP-40 in the reaction solution is 0.01-1%; Preferably, the content of dATP in the reaction solution is 0.2-8mM, more preferably 2-4mM; Preferably, the content of dNTP in the reaction solution is 0.02-2mM, more preferably 0.02-1mM; Preferably, the content of ATP in the reaction solution is 5-15mM. 9. A method for constructing a sequencing library, characterized in that the method comprises: adding target nucleic acid to the enzyme composition according to any one of claims 1-3 and the enzyme composition according to any one of claims 4-8 Incubate in the mixture of reaction solution to carry out fragmentation of target nucleic acid, end repair, 5' phosphorylation and 3' A tailing. 10. A kit for constructing a sequencing library, characterized in that the kit includes the enzyme composition according to any one of claims 1-3 or the reaction solution according to any one of claims 4-8 ; Preferably, the kit also includes sequencing adapters and ligation reagents.

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