CN114196661A - Recombinant topoisomerase and application thereof in constructing sequencing library - Google Patents
Recombinant topoisomerase and application thereof in constructing sequencing library Download PDFInfo
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Abstract
The invention discloses a recombinant topoisomerase and application thereof in constructing a sequencing library. The invention firstly discloses a recombinant topoisomerase with an amino acid sequence shown as SEQ ID NO. 1. The invention further discloses application of the recombinant topoisomerase in construction of a sequencing library. The invention creatively applies the topoisomerase to an enzyme-cutting DNA library building system, so that the treatment efficiency of fragmenting, end repairing and 3' tail adding of a DNA sample is higher, and the balance is easier to achieve; in addition, the DNA melting and cutting actions of the recombinant topoisomerase solve the problems that the end repair is not thorough due to the formation of double chains by the quick annealing of single chains in the enzyme digestion reaction in the construction process of the sequencing library, the GC distribution of a DNA high GC area in the sequencing is unbalanced, and the conversion efficiency of the library is low when the initial amount of a sample is low, improve the yield and the quality of the sequencing library, and have wide applicability and convenient operability.
Description
Technical Field
The invention relates to the field of biotechnology. More particularly, it relates to a recombinant topoisomerase and its application in the construction of sequencing library.
Background
With the rapid development of molecular diagnostic technology, the application fields of the molecular diagnostic technology are more and more, and particularly with the development of Metagenomic Next-Generation Sequencing (mNGS), the demand of people for rapid DNA library construction is more and more urgent. In general, NGS pooling involves (1) nucleic acid (DNA or RNA) fragmentation; (2) screening the fragmentation size; (3) adding linkers to the fragmented nucleic acids; (4) sequencing (Behjati S, Tarpey PS. What is next generation sequencing. As the first step of NGS library construction, which is also the most critical step of nucleic acid fragmentation, two methods of mechanical ultrasonication and enzymatic cleavage are mainly used at present (Nelly Sapojnikova, Nino Asatiani, Tamar Kartvelisvili, Lali Asansivili, vitally Zinkevich, Irina Bogdarina, Julian Mitchell, Abdulmohsen Al-Humam, A contrast of DNA fragmentation methods-Applications for the biochip technology, Journal of Biotechnology, Volume 256,2017, Pages 1-5.). Because the instrument interrupted by mechanical ultrasound is expensive, occupies a large area, has low flux, is complicated to operate and long in time, and the required samples have large quantity (the loss of micro-sample fragmentation is large), the rapid processing efficiency of the DNA library on multiple samples is seriously limited, and the popularization of the DNA library in the medical field is hindered. The use of DNA fragmentation enzyme (i.e. enzyme digestion) effectively solves the problem of mechanical ultrasound disruption: the wide template input amount range (100pg-1 mug), controllable DNA fragmentation in a PCR tube, and the requirements of high throughput, simple operation and low DNA input amount of DNA library construction are met.
The existing enzyme cutting method DNA library construction is based on the following principle: 1>Random nicking of DNA using nicking enzyme, while recognition and cleavage of nick sites using T7 endonucleoclean I; 2>Using DNase I or dsDNase at very low dosages in Mg2+And Mn2+Fragmenting dsDNA under the action of the action; 3>Fragmenting dsDNA using a combination of multiple restriction enzymes; 4>Strand displacement energy with polymerase using VVN or DNase I or dsDNase at very low dosagesForce to fragment. However, both methods have a preference for fragmentation (limited by the preference for nucleases), and heterogeneity of GC distribution in high GC regions (single strands formed after cleavage in high GC regions tend to anneal to double strands); certain influences are caused for SV and SNP determination and identification of the library (Chen Y-C, Liu T, Yu C-H, Chiang T-Y, Hwang C-C (2013) Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome ploS ONE 8(4): 62e 856.).
Topoisomerase catalyzes a coupling reaction of DNA strand breakage and binding, and can cut off DNA of one strand or two strands (nick at a position to be knotted or knotted) and restore the ligation function (Natassja G Bush, Katherine Evans-Roberts, Anthony Maxwell. DNA topoisomerases. EcoSul plus. 2015; 6(2) doi: 10.1128/ecoalplus. ESP-0010-. However, in addition to cleaving, topoisomerase can also reset the linkage, a function which is not required.
Therefore, it is desirable to provide a novel topoisomerase for use in sequencing library construction.
Disclosure of Invention
The first purpose of the invention is to provide a recombinant topoisomerase, which is combined with the existing enzyme digestion reaction liquid for constructing a sequencing library, and can effectively improve the yield and the sequencing data quality of the library constructed by the enzyme digestion sequencing library.
The second object of the present invention is to provide an enzymatic cleavage reaction solution containing the above recombinant topoisomerase.
The third object of the present invention is to provide a kit comprising the above recombinant topoisomerase or the above enzyme-cleaved reaction solution.
The fourth purpose of the invention is to provide the application of the recombinant topoisomerase, the enzyme digestion reaction solution or the kit in constructing a sequencing library.
The fifth purpose of the invention is to provide a construction method of a sequencing library.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a recombinant topoisomerase, wherein the amino acid sequence of the recombinant topoisomerase is shown in SEQ ID NO. 1.
The recombinant topoisomerase is obtained by mutating and modifying Escherichia coli type I topoisomerase (the amino acid sequence of which is shown as SEQ ID No. 6), so that the reset connection function of the topoisomerase is greatly reduced/deleted, the DNA cutting function of the topoisomerase is acted on the construction of a sequencing library to participate in DNA fragmentation, terminal modification and 3' A tail addition reaction, the heterogeneity of GC distribution in the traditional library construction is greatly improved, and the preference of GC distribution in sequencing analysis is more consistent in a high GC area.
In a second aspect, the invention provides an enzyme digestion reaction solution, which comprises a fragmentation enzyme mixed solution, wherein the fragmentation enzyme mixed solution comprises the recombinant topoisomerase.
Further, the fragmentation enzyme mixture also comprises endonuclease and DNA polymerase.
The recombinant topoisomerase in the enzyme digestion reaction liquid can be combined with different endonucleases (used for cutting or nicking a DNA double strand), and comprises any one or combination of two of double-strand DNA nuclease and ATP-dependent nuclease; the recombinant topoisomerase does not influence the enzyme cutting effect of endonuclease.
The recombinant topoisomerase in the enzyme digestion reaction liquid can be combined with different DNA polymerases (used for carrying out end repair on the cut nucleic acid or carrying out A tail adding reaction on the 3' end of the cut nucleic acid), and comprises any one or the combination of two of low-temperature DNA polymerase and heat-resistant DNA polymerase; the low-temperature DNA polymerase comprises any one or the combination of Phi29DNA polymerase and T4 DNA polymerase large fragment Klenow; the heat-resistant DNA polymerase comprises any one or the combination of Bst II DNA polymerase and Taq DNA polymerase large fragment Klenow; the recombinant topoisomerase does not affect the polymerization effect of the DNA polymerase.
Further, the fragmentation enzyme mixture also comprises an auxiliary protein, and the auxiliary protein is used for regulating the cutting rate of the endonuclease and the end repairing rate of the DNA polymerase.
The recombinant topoisomerase in the enzyme digestion reaction liquid can be combined with a plurality of auxiliary proteins, wherein the auxiliary proteins comprise any one or the combination of two of recombinant albumin (BSA substitute) and single-chain binding protein (SSB); the recombinant topoisomerase does not affect the regulatory function of these helper proteins.
In a specific embodiment of the present invention, the fragmentation enzyme mixture is composed of the above-mentioned recombinant topoisomerase (amino acid sequence shown in SEQ ID NO. 1), ATP-dependent nuclease (amino acid sequence shown in SEQ ID NO. 2), Phi29DNA polymerase (TransGen, LP101), Bst II DNA polymerase (TransGen, LP301), SSB (amino acid sequence shown in SEQ ID NO. 4), recombinant albumin (amino acid sequence shown in SEQ ID NO. 3), dsDNase (TransGen, LD101), Tris-HCl, KCl, Tween 20, DTT and glycerol.
In a preferred embodiment of the invention, the final concentration of the recombinant topoisomerase in the fragmentation enzyme mixture is from 4 ng/. mu.l to 15 ng/. mu.l; the final concentration of the ATP-dependent nuclease in the fragmentation enzyme mixture is 0.4 ng/. mu.l to 0.8 ng/. mu.l; the final concentration of the Phi29DNA polymerase in the fragmentation enzyme mixture is 15 ng/mu l-25 ng/mu l; the final concentration of the Bst II DNA polymerase in the fragmentation enzyme mixture is 7.5 ng/mu l-15 ng/mu l; the final concentration of the SSB in the fragmentation enzyme mixture is 120 ng/mu l-300 ng/mu l; the final concentration of the recombinant albumin in the fragmentation enzyme mixed liquor is 300 ng/mu l-700 ng/mu l; the final concentration of the dsDNase in the fragmentation enzyme mixture is 0.6 ng/mu l-1.0 ng/mu l; the final concentration of Tris-HCl in the fragmentation enzyme mixture is 40 mM-60 mM; the final concentration of the KCl in the fragmentation enzyme mixture is 15mM-40 mM; the final concentration of the Tween 20 in the fragmentation enzyme mixed solution is 0.25-0.75% (V/V); the final concentration of the DTT in the fragmentation enzyme mixture is 0.5mM-2 mM; the final concentration of the glycerol in the fragmentation enzyme mixture is 40-60% (V/V).
In a more preferred embodiment of the invention, the final concentration of the recombinant topoisomerase in the fragmentation enzyme mixture is 11.0 ng/. mu.l; the final concentration of the ATP-dependent nuclease in the fragmentation enzyme mixture is 0.6 ng/. mu.l; the final concentration of the Phi29DNA polymerase in the fragmentation enzyme mixture is 20 ng/. mu.l; the final concentration of the Bst II DNA polymerase in the fragmentation enzyme mixture is 10 ng/. mu.l; the final concentration of the SSB in the fragmentation enzyme mixture is 200 ng/. mu.l; the final concentration of the recombinant albumin in the fragmentation enzyme mixed liquor is 500 ng/mu l; the final concentration of the dsDNase in the fragmentation enzyme mixture is 0.8 ng/. mu.l; the final concentration of Tris-HCl in the fragmentation enzyme mixture is 50 mM; the final concentration of the KCl in the fragmentation enzyme mixture is 25 mM; the final concentration of the Tween 20 in the fragmentation enzyme mixture is 0.5% (V/V); the final concentration of the DTT in the fragmentation enzyme mixture is 1 mM; the final concentration of glycerol in the fragmentation enzyme mixture was 50% (V/V).
The recombinant topoisomerase can be integrated with the existing endonuclease used for cutting or nicking a DNA double strand and DNA polymerase used for carrying out end repair on the cut nucleic acid or carrying out A-tail addition reaction on the 3' end of the cut nucleic acid in a reaction system, and the self-hybridized double-strand DNA formed by the exposed single-strand DNA in a cut nucleic acid sample is melted, so that the preference of a high GC part in the sequencing of the constructed sequencing library is effectively avoided.
Further, the enzyme digestion reaction solution also comprises an enzyme digestion reaction buffer solution. The enzyme digestion buffer solution can be the common enzyme digestion buffer solution in the prior art. The enzyme digestion reaction buffer solution comprises metal cations, a substrate and a buffer medium. The metal cation comprises Mg2+、K+And NH3 +Any one or more combinations thereof, such as MgCl2KAc; the substrate comprises any one or more of dNTPs, dATP and ATP, such as dNTPs, dATP and ATP; the buffer medium comprises any one or combination of more of 2-morpholinoethanesulfonic acid (MES), acetic acid (Ac), and Tris, such as Tris-acetic acid; the enzymeThe cleavage reaction buffer may perform the melting action without inhibiting or hindering the fragmentation enzyme mixture described above.
In a specific embodiment of the invention, the enzyme digestion buffer consists of Tris-acetic acid, MgCl2KAc, dNTPs, dATP and ATP.
In a preferred embodiment of the invention, the final concentration of Tris-acetate in the cleavage reaction buffer is between 200mM and 400 mM; said MgCl2The final concentration in the enzyme digestion reaction buffer solution is 75mM-125 mM; the KAc in the enzyme digestion reaction buffer solution is 100mM-300 mM; the final concentration of the dNTPs in the enzyme digestion reaction buffer solution is 1.5mM-3 mM; the final concentration of the dATP in the enzyme digestion reaction buffer solution is 3mM-7 mM; the final concentration of ATP in the enzyme reaction buffer is 20mM-35 mM.
In a more preferred embodiment of the present invention, the final concentration of Tris-acetate in the cleavage reaction buffer is 300 mM; said MgCl2The final concentration in the enzyme digestion reaction buffer solution is 100 mM; the KAc in the enzyme digestion reaction buffer solution is 200 mM; the final concentration of the dNTPs in the enzyme digestion reaction buffer solution is 2 mM; the final concentration of the dATP in the enzyme digestion reaction buffer solution is 5 mM; the final concentration of ATP in the cleavage reaction buffer was 25 mM.
In a specific embodiment of the present invention, the pH of the digestion buffer is 7.5-8.5. In a preferred embodiment of the present invention, the pH of the digestion buffer is 8.
The recombinant topoisomerase has stronger adaptability to the combination of the metal cation type and concentration in the enzyme digestion reaction buffer solution and the type and concentration of the buffer medium within a certain range, has no influence on the activity of endonuclease and DNA polymerase, and performs a melting function to ensure that a palindromic structure region of special high GC is easier to detect, thereby being more beneficial to base balance and avoiding GC separation.
In a third aspect, the invention provides a kit, which comprises the recombinant topoisomerase or the enzyme digestion reaction solution.
Further, the kit also comprises a joint connection reaction liquid, wherein the joint connection reaction liquid comprises a joint ligase mixed liquid and a joint connection reaction buffer liquid.
The mixed solution of the adaptor ligase in the kit can be the mixed solution of the adaptor ligase commonly used in the prior art. In the present invention, the adaptor ligase mixture comprises a polynucleotide kinase and a ligase; the polynucleotide kinase may be a T4 polynucleotide kinase (T4 PNK) that 5' phosphorylates cleaved nucleic acids; the ligase may be T4 DNA ligase to ligate the linker.
In a specific embodiment of the invention, the adaptor ligase mixture consists of T4 DNA ligase, T4 PNK, Tris-Ac, Tween 20, DTT and glycerol.
In a preferred embodiment of the present invention, the final concentration of the T4 DNA ligase in the adaptor ligase mixture is 600 ng/. mu.l-1. mu.g/. mu.l; the final concentration of the T4 PNK in the adaptor ligase mixed solution is 30 ng/mu l-100 ng/mu l; the final concentration of the Tris-Ac in the adaptor ligase mixture is 30mM-100 mM; the final concentration of the Tween 20 in the adaptor ligase mixed solution is 0.25-0.7% (V/V); the final concentration of the DTT in the joint ligase mixed solution is 0.5mM-2 mM; the final concentration of the glycerol in the adaptor ligase mixed solution is 40-60% (V/V).
In a more preferred embodiment of the present invention, the final concentration of the T4 DNA ligase in the adaptor ligase mixture is 800 ng/. mu.l; the final concentration of the T4 PNK in the adaptor ligase mixed solution is 80 ng/. mu.l; the final concentration of Tris-Ac in the adaptor ligase mixture is 50 mM; the final concentration of the Tween 20 in the adaptor ligase mixture is 0.5% (V/V); the final concentration of the DTT in the joint ligase mixed solution is 1 mM; the final concentration of glycerol in the adaptor ligase mixture was 50% (V/V).
The linker ligation reaction buffer in the present invention may be a linker ligation reaction buffer commonly used in the prior art; in a specific embodiment of the invention, the linker Ligation reaction Buffer is Adapter Ligation Buffer in TransGen # KP201TransNGS DNA Library Prep Kit for Illumina.
Further, the kit also comprises any one or more of a sequencing joint, a PCR amplification enrichment reagent and a purification reagent.
In a fourth aspect, the invention provides an application of the recombinant topoisomerase, the enzyme digestion reaction solution or the kit in constructing a sequencing library or an application in preparing a reagent for constructing a sequencing library.
In a fifth aspect, the present invention provides a method for constructing a sequencing library, the method comprising:
fragmenting, end repairing and 3' adding A tail to target DNA by using enzyme digestion reaction liquid containing the recombinant topoisomerase, the enzyme digestion reaction liquid or the enzyme digestion reaction liquid in the kit to obtain a fragmentation product;
carrying out 5' phosphorylation and linker ligation reaction on the fragmentation product to obtain a phosphorylation and linker ligation product;
and performing PCR amplification and enrichment on the phosphorylation and adaptor connection products to obtain a sequencing library.
Further, the reaction conditions of fragmentation, end repair and 3' tail A addition are that incubation is carried out for 5-30 minutes at 32 ℃ or 37 ℃, and then incubation is carried out for 20-30 minutes at 65 ℃.
Further, the 5 'phosphorylation and linker ligation reaction is performed in a linker ligation reaction solution in the kit, and the reaction conditions of the 5' phosphorylation and linker ligation reaction are incubation at 25 ℃ for 15-20 minutes.
Further, the amount of the target DNA to be added is 100pg to 1. mu.g.
Further, the construction method further comprises a step of purifying the obtained product after performing a linker ligation reaction.
Further, the construction method also comprises the step of purifying the obtained product after PCR amplification and enrichment.
The invention has the following beneficial effects:
1) according to the invention, topoisomerase is creatively applied to an enzyme cutting method DNA library construction system, in the sequencing library construction process, recombinant topoisomerase is added into a fragmentation enzyme mixed solution and is mutually matched with metal cations and a buffer medium in an enzyme cutting reaction buffer solution, and DNA sample fragmentation, terminal repair and 3 'A tail addition are integrated into one-step reaction, so that the cutting, terminal repair and 3' A tail addition processing efficiencies of the enzyme cutting reaction solution on a nucleic acid sample are higher, and the balance is more easily achieved; in addition, the DNA melting and cutting actions of the recombinant topoisomerase solve the problems that the end repair is not thorough due to the formation of double chains by the quick annealing of single chains in the enzyme digestion reaction in the construction process of the sequencing library, the GC distribution of a DNA high GC area in the sequencing is unbalanced, and the conversion efficiency of the library is low when the initial amount of a sample is low, improve the yield and the quality of the sequencing library, and have wide applicability and convenient operability.
2) In the invention, 5' phosphorylation reacts with the joint connection system in the process of constructing a sequencing library, so that the joint which is subjected to phosphorylation failure due to repeated freeze thawing is subjected to phosphorylation again, the joint connection efficiency of the joint is improved, and the conversion rate of the library and the success rate of library construction are further improved on the basis of not changing the process of constructing the library by an enzyme cutting method DNA.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a 2100 peak profile of the sequencing library constructed in combination 1;
FIG. 2 is a 2100 peak profile of the sequencing library constructed in combination 2;
FIG. 3 is a 2100 peak profile of the sequencing library constructed in combination 3;
FIG. 4 is a 2100 peak profile of the sequencing library constructed in combination 5;
FIG. 5 is a 2100 peak profile of the sequencing library constructed in combination 6;
FIG. 6 is a 2100 peak profile of the sequencing library constructed in combination 7;
FIG. 7 is a GC content distribution and depth distribution graph after using combinations 5, 6, 7 to build libraries with different DNA input amounts; wherein the amount of DNA introduced into A was 100pg, the amount of DNA introduced into B was 1ng, the amount of DNA introduced into C was 10ng, the amount of DNA introduced into D was 100ng, and the amount of DNA introduced into E was 1. mu.g.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Wherein, the sources of part of the reagents in the examples are as follows:
ATP-dependent nucleases: the amino acid sequence is shown as SEQ ID NO. 2;
recombinant albumin (BSA substitute): the amino acid sequence is shown as SEQ ID NO. 3;
SSB (single chain binding protein): the amino acid sequence is shown as SEQ ID NO. 4;
taq DNA polymerase large fragment Klenow: the amino acid sequence is shown as SEQ ID NO. 5;
dsDNase：TransGen，LD101；
phi29DNA polymerase: TransGen, LP 101;
bst II DNA polymerase: TransGen, LP 301;
t4 DNA ligase: TransGen, LL 101;
T4 PNK：TransGen，LK101。
example 1 construction of recombinant topoisomerase
The recombinant topoisomerase is a novel topoisomerase reconstructed according to Escherichia coli type I topoisomerase (the amino acid sequence is shown as SEQ ID NO. 6), and specifically comprises: the Escherichia coli I type topoisomerase is subjected to mutation transformation of enzyme evolution, the topoisomerase with the reset connection function greatly reduced/deleted is obtained by screening, and the DNA cutting function of the topoisomerase is acted on the library construction of DNA, so that the heterogeneity of GC distribution of the traditional library construction is greatly improved.
Finally, the amino acid sequence of the obtained recombinant topoisomerase is shown in SEQ ID NO. 1.
EXAMPLE 2 preparation of enzyme digestion reaction solution
For better reaction of functions of the topoisomerase, the fragmentation enzyme mixed liquor in the enzyme digestion reaction liquor is prepared into fragmentation enzyme mixed liquor I without the topoisomerase, fragmentation enzyme mixed liquor II and III with the recombinant topoisomerase in the embodiment 1 and fragmentation mixed liquor IV with the escherichia coli type I topoisomerase; the enzyme digestion reaction buffer solution is used for the preparation of the enzyme digestion reaction solution.
1. Preparing a fragmentation enzyme mixed solution:
the components of the fragmentation enzyme mixed liquor I and the final concentration of each component in the fragmentation enzyme mixed liquor I are as follows: ATP dependent nuclease (0.6 ng/. mu.l), dsDNase (0.8 ng/. mu.l), recombinant albumin (BSA substitute, 400 ng/. mu.l), Phi29DNA polymerase (20 ng/. mu.l), Taq DNA polymerase large fragment Klenow (10 ng/. mu.l), SSB (single stranded binding protein, 200 ng/. mu.l), Tris-HCl (pH8.0, 50mM), KCl (25mM), Tween 20(V/V, 0.5%), DTT (1mM), glycerol (V/V, 50%).
The components of the fragmentation enzyme mixed liquor II and the final concentration of each component in the fragmentation enzyme mixed liquor II are as follows: example 1 the amino acid sequence of the recombinant topoisomerase shown in SEQ ID No.1 (5.5 ng/. mu.l), ATP dependent nuclease (0.6 ng/. mu.l), dsDNase (0.8 ng/. mu.l), recombinant albumin (BSA substitute, 500 ng/. mu.l), Phi29DNA polymerase (20 ng/. mu.l), Bst II DNA polymerase (10 ng/. mu.l), SSB (single-stranded binding protein, 200 ng/. mu.l), Tris-HCl (pH8.0, 50mM), KCl (25mM), Tween 20(V/V, 0.5%), DTT (1mM), glycerol (V/V, 50%).
The components of the fragmentation enzyme mixed liquor III and the final concentration of each component in the fragmentation enzyme mixed liquor III are as follows: example 1 the amino acid sequence of the recombinant topoisomerase shown in SEQ ID No.1 (11.0 ng/. mu.l), ATP dependent nuclease (0.6 ng/. mu.l), dsDNase (0.8 ng/. mu.l), recombinant albumin (BSA substitute, 400 ng/. mu.l), Phi29DNA polymerase (20 ng/. mu.l), Bst II DNA polymerase (10 ng/. mu.l), SSB (single-stranded binding protein, 200 ng/. mu.l), Tris-HCl (pH8.0, 50mM), KCl (25mM), Tween 20(V/V, 0.5%), DTT (1mM), glycerol (V/V, 50%).
The components of the fragmentation enzyme mixed liquor IV and the final concentration of each component in the fragmentation enzyme mixed liquor IV are as follows: the amino acid sequence described in example 1 is shown in SEQ ID NO.6 for E.coli type I topoisomerase (11.0 ng/. mu.l), ATP dependent nuclease (0.6 ng/. mu.l), dsDNase (0.8 ng/. mu.l), recombinant albumin (BSA substitute, 400 ng/. mu.l), Phi29DNA polymerase (20 ng/. mu.l), Bst II DNA polymerase (10 ng/. mu.l), SSB (single stranded binding protein, 200 ng/. mu.l), Tris-HCl (pH8.0, 50mM), KCl (25mM), Tween 20(V/V, 0.5%), DTT (1mM), glycerol (V/V, 50%).
2. Preparation of enzyme digestion reaction buffer solution:
the components of the enzyme digestion reaction buffer solution and the final concentration of each component in the enzyme digestion reaction buffer solution are as follows: tris-acetic acid (pH8.0, 300mM), MgCl2(100mM)，KAc(200mM)，dNPTs(2mM)，dATP(5mM)，ATP(25mM)。
3. Preparation of enzyme digestion reaction liquid:
enzyme digestion reaction solution 1: the fragmentation enzyme mixed liquor I + enzyme digestion reaction buffer solution;
and (3) enzyme digestion reaction liquid 2: fragmenting enzyme mixed liquor II + enzyme digestion reaction buffer solution;
enzyme digestion reaction solution 3: and (3) fragmenting enzyme mixed liquor III + enzyme digestion reaction buffer solution.
Enzyme digestion reaction solution 4: and (4) fragmenting enzyme mixed liquor IV + enzyme digestion reaction buffer solution.
EXAMPLE 3 preparation of linker ligation reaction solution
The linker ligation reaction solution comprises two parts: the joint ligase mixed solution and the joint ligation reaction buffer solution.
1. Preparing a joint ligase mixed solution:
the components of the adaptor ligase mixed solution 1 and the final concentration of each component in the adaptor ligase mixed solution 1 are as follows: t4 DNA ligase (800 ng/. mu.l), T4 PNK (40 ng/. mu.l), Tris-Ac (pH8.0, 50mM), Tween 20(V/V, 0.5%), DTT (1mM), glycerol (V/V, 50%).
The components of the adaptor ligase mixed solution 2 and the final concentration of each component in the adaptor ligase mixed solution 2 are as follows: t4 DNA ligase (800 ng/. mu.l), T4 PNK (80 ng/. mu.l), Tris-Ac (pH8.0, 50mM), Tween 20(V/V, 0.5%), DTT (1mM), glycerol (V/V, 50%).
2. Preparing a joint connection reaction buffer solution:
linker ligation reaction buffer: an Adapter Ligation Buffer in the TransGen # KP201TransNGS DNA Library Prep Kit for Illumina was used.
3. Preparing a joint connection reaction solution:
the joint connection reaction solution is composed of a joint connection enzyme mixed solution and a joint connection reaction buffer solution:
linker-linking reaction solution 1: the joint ligase mixed solution 1+ joint ligation reaction buffer solution;
linker-linking reaction solution 2: and (3) joint ligase mixed solution 2+ joint ligation reaction buffer solution.
Example 4 construction of DNA sequencing library
In this example, HeLa cell gDNA was used as a template, the initial input amounts were 100pg, 1ng, 10ng, 100ng and 1. mu.g, the gDNA was fragmented, end-repaired and 3' tailed with A by using different digestion solutions of example 1, and then linker ligation was performed by using different linker ligation solutions of example 2 (linker # KP 201)
In DNA Library Prep Kit for Illumina
Adapter for
Or Adapter in KI 401) and then purifying the adaptor ligation DNA Beads using TransGen # EC401 MagicPure Size Selection using TransGen # KP201
DNA Library Prep Kit for
In (1)
The purified ligation products were PCR amplified and enriched by Library Amplification Supermix (2 ×) and i5/i7 Primer in TransGen # KI251, and finally the construction of DNA sequencing Library was completed.
Fragmentation, end repair and 3' dA tailing of HeLa cell gDNA
The reaction systems of fragmentation, end repair and 3' dA tail addition of Hela cell gDNA are shown in Table 1, wherein one of the fragmentation enzyme mixtures I-III in example 1 is adopted as the formula of the fragmentation enzyme mixture, the digestion reaction buffer in example 1 is adopted as the digestion reaction buffer, and the input amount of Hela cell gDNA (hereinafter referred to as DNA) is X (corresponding to 100pg, 1ng, 10ng, 100ng and 1 μ g respectively).
TABLE 1 fragmentation, end repair and 3' dA-tailed reaction System
And (3) uniformly mixing by using a pipette, and then incubating for 12 minutes at 37 ℃ and 30 minutes at 65 ℃ according to reaction conditions to carry out DNA enzyme digestion (fragmentation), end repair and 3' A tail addition to obtain a fragmentation product.
2. 5' phosphorylation, linker ligation and purification of fragmentation products
After fragmentation, end repair and 3 'dA tail addition, the fragmentation product needs to be 5' phosphorylated, linker ligated and purified.
Using TransGen # KP201
Adapter Dilution Buffer pair # KP201 in DNA Library Prep Kit for Illumina
In DNA Library Prep Kit for Illumina
Adapter for
Linker dilutions were performed to obtain linker concentrations, as shown in Table 2.
TABLE 2 linker dilution ratios for different DNA inputs
| DNA input amount | Dilution factor of joint | Corresponding linker concentration after dilution |
| 100ng＜x≤1μg | Is not diluted | 16μM |
| 25ng＜x≤100ng | Diluting by 2 times | 8μM |
| 5ng＜x≤25ng | Diluting by 10 times | 1.6μM |
| 100pg≤x≤5ng | Diluting by 25 times | 0.6μM |
The 5' phosphorylation and linker ligation reaction system is shown in Table 3, wherein the linker ligase mixture was prepared using either one of the linker ligase mixtures 1 and 2 of example 2, and the linker ligation buffer was prepared using the linker ligation buffer of example 2.
TABLE 35' phosphorylation and linker ligation reaction systems
The mixture was pipetted and mixed and then incubated at 25 ℃ for 15 minutes for 5' phosphorylation and adaptor ligation reactions (long adaptors recommended 20 ℃ ligation reaction for 15 minutes) under reaction conditions to give phosphorylated and adaptor ligated products.
The phosphorylated and linker ligation products were purified using 80. mu.L TransGen # EC401 MagicPure Size Selection DNA Beads, elution volume was 20/23. mu.L, to give purified phosphorylated and linker ligation products.
3. PCR amplification enrichment of purified adaptor ligation products
Using TransGen # KP201
Library Amplification Supermix (Library Amplification reaction solution) and TransGen # KI251
UDI Primers(96)Kit for
(i5/i7 Pirmer) PCR amplification enrichment was performed on the purified adaptor ligation products, and the PCR amplification enrichment system is shown in Table 4.
TABLE 4 PCR amplification enrichment System
| Components | Dosage of |
| Phosphorylated and linker ligation products | 20μL |
| Library amplification reaction solution | 25μL |
| i5 Primer | 2.5μL |
| i7 Primer | 2.5μL |
And (3) uniformly mixing by using a pipette, and then carrying out PCR amplification and enrichment according to reaction conditions to obtain an amplification product.
The PCR amplification conditions were as follows:
TABLE 5 amplification cycle numbers for different DNA inputs
| DNA input amount | Number of amplification cycles X |
| 100pg | 17 |
| 1ng | 15 |
| 10ng | 9 |
| 100ng | 4 |
| |
2 |
After the amplification is finished, 50 mu L of TransGen # EC401 MagicPure Size Selection DNA Beads are used for purifying the amplification product to obtain a DNA sequencing library; quantifying DNA sequencing library yield using Qubit; the length distribution of the DNA sequencing library was detected using the Agilent 2100 DNA 1000 chip, and the results are shown in Table 6 and FIGS. 1-6, which shows that the combination of each set of the enzymatic cleavage reaction solution and the linker ligation reaction solution can achieve effective cleavage (fragmentation), end repair, 3 'dA tail addition, 5' phosphorylation and linker ligation of DNAs with different input amounts.
List of different combinations:
combination 1: the enzyme digestion reaction solution 1+ joint is connected with the reaction solution 1; and (3) combination 2: the enzyme digestion reaction solution 2+ joint is connected with the reaction solution 1; and (3) combination: the enzyme digestion reaction solution 3+ joint is connected with the reaction solution 1; and (4) combination: the enzyme digestion reaction solution 4+ joint is connected with the reaction solution 1; and (3) combination 5: the enzyme digestion reaction solution 1+ joint is connected with the reaction solution 2; and (4) combination 6: the enzyme digestion reaction solution 2+ joint is connected with the reaction solution 2; and (3) combination 7: the enzyme digestion reaction solution 3+ joint is connected with the reaction solution 2; and (4) combination 8: the enzyme digestion reaction solution 4+ joint is connected with the reaction solution 2.
TABLE 6 production of libraries constructed in different combinations: (unit: ng)
| DNA | Combination | 1 | |
|
|
|
Combination 7 |
| 100pg | 676.3 | 777.7 | 894.4 | 689.8 | 786.5 | 914.7 | |
| 1ng | 882.5 | 1017.5 | 1173.2 | 917.8 | 1028.1 | 1197.8 | |
| 10ng | 1011.2 | 1167.9 | 1349.0 | 1071.9 | 1180.1 | 1377.1 | |
| 100ng | 1355.2 | 1568.0 | 1814.1 | 1463.6 | 1584.2 | 1852.0 | |
| 1μg | 1566.4 | 1815.5 | 2104.1 | 1723.0 | 1834.3 | 2147.9 |
Where pool 4 and pool 8 failed to build a library, there was no data.
By analysis, it is known that: pool building fails for pool 4 and pool 8. DNA sequencing libraries are successfully constructed from combinations 1 to 3 and from combinations 5 to 7, and the peak types of the libraries are not very different, which shows that the addition of the recombinant topoisomerase has little influence on the peak types; the yield of DNA sequencing libraries constructed in combination 1 versus combination 5, combination 2 versus combination 6, and combination 3 versus combination 7 increased somewhat, indicating that the yield of the library increased somewhat with increasing T4 PNK; the lower yields of combination 1 relative to combinations 2 and 3 and combination 5 relative to combinations 6 and 7 indicate that the addition of the recombinant topoisomerase of the invention results in a significant increase in the yield of the DNA sequencing library.
Example 5 GC distribution analysis
As is clear from the DNA sequencing library yields and peak patterns of example 4, the yields of the DNA sequencing libraries constructed in combination 5, combination 6 and combination 7 were higher than those of combination 1, combination 2 and combination 3, respectively, so that the DNA sequencing libraries constructed in combination 5, combination 6 and combination 7 were sorted using TransGen # EC401 MagicPure Size Selection DNA Beads (0.65 × +0.2 ×) and sent to a third party sequencing company for sequencing and analysis, and the analysis results are shown in Table 7.
TABLE 7 sequencing data quantity and Mass analysis results
Picard (version: 1.119, parameter: CollectGcBiasMetrics. jar) was used to make statistics of the GC content distribution in the genome and the GC distribution in the alignment results, and to plot the results, it was possible to know whether there was a GC preference in the sequencing data (generally, there was a certain GC preference in both the low GC content and high GC content portions, and the closer to 1 the relative coverage, the smaller the GC preference).
The GC content distribution and depth profile of the pools prepared using combinations of DNA input amounts 5, 6 and 7 are shown in A-E in FIG. 7 (the abscissa represents GC content (the genome is calculated using 100bp as a window), and the ordinate represents the relative coverage of a specific base content (left) and the window ratio of the occupied genome (right)).
As can be seen from FIG. 7, the GC distributions in the high GC regions are more balanced after the recombinant topoisomerase of example 1 of the present invention is added in combination 6 and 7 than in combination 5.
In conclusion, the addition of the recombinant topoisomerase does not affect the enzyme digestion effect of the enzyme digestion reaction solution on DNA, but obviously improves the yield of the constructed DNA sequencing library, and obviously improves the deviation of GC distribution in sequencing data analysis.
Therefore, the addition of the recombinant topoisomerase improves the library construction quality of the DNA sequencing library by the enzyme cutting method, and the subsequent sequencing data analysis is greatly ensured.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
SEQUENCE LISTING
<110> Beijing Quanjin Biotechnology Ltd
<120> recombinant topoisomerase and application thereof in constructing sequencing library
<130> JLP21I1306
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<170> PatentIn version 3.5
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85 90 95
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290 295 300
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305 310 315 320
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385 390 395 400
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405 410 415
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420 425 430
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435 440 445
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450 455 460
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465 470 475 480
Ser Leu Val Lys Glu Leu Glu Lys Arg Gly Ile Gly Arg Pro Ser Thr
485 490 495
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500 505 510
Glu Asn Arg Arg Phe Tyr Ala Glu Lys Met Gly Glu Ile Val Thr Asp
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Arg Leu Glu Glu Asn Phe Arg Glu Leu Met Asn Tyr Asp Phe Thr Ala
530 535 540
Gln Met Glu Asn Ser Leu Asp Gln Val Ala Asn His Glu Ala Glu Trp
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565 570 575
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Val Leu Thr Ser Ile Asp Cys Pro Thr Cys Gly Arg Lys Met Gly Ile
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Arg Thr Ala Ser Thr Gly Val Phe Leu Gly Cys Ser Gly Tyr Ala Leu
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Glu Asp Pro Val Pro Leu Pro Glu Leu Pro Cys Glu Lys Ser Asp Ala
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Tyr Phe Val Leu Arg Asp Gly Ala Ala Gly Val Phe Leu Ala Ala Asn
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Thr Phe Pro Lys Ser Arg Glu Thr Arg Ala Pro Leu Val Glu Glu Leu
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Tyr Arg Phe Arg Asp Arg Leu Pro Glu Lys Leu Arg Tyr Leu Ala Asp
805 810 815
Ala Pro Gln Gln Asp Pro Glu Gly Asn Lys Thr Met Val Arg Phe Ser
820 825 830
Arg Lys Thr Lys Gln Gln Tyr Val Ser Ser Glu Lys Asp Gly Lys Ala
835 840 845
Thr Gly Trp Ser Ala Phe Tyr Val Asp Gly Lys Trp Val Glu Gly Lys
850 855 860
Lys
865
Claims (10)
1. A recombinant topoisomerase is characterized in that the amino acid sequence of the recombinant topoisomerase is shown as SEQ ID NO. 1.
2. An enzyme digestion reaction solution, comprising a fragmentation enzyme mixture, wherein the fragmentation enzyme mixture comprises the recombinant topoisomerase of claim 1.
3. The enzyme digestion reaction solution according to claim 2, wherein the fragmentation enzyme mixture solution further comprises an endonuclease and a DNA polymerase;
preferably, the endonuclease comprises any one or a combination of two of a double-stranded DNA nuclease and an ATP-dependent nuclease; the DNA polymerase comprises any one or combination of two of low-temperature DNA polymerase and heat-resistant DNA polymerase;
more preferably, the low-temperature DNA polymerase comprises any one or the combination of two of Phi29DNA polymerase and T4 DNA polymerase large fragment Klenow; the heat-resistant DNA polymerase comprises any one or the combination of Bst II DNA polymerase and Taq DNA polymerase large fragment Klenow.
4. The enzyme digestion reaction solution according to claim 3, wherein the fragmentation enzyme mixture solution further comprises an accessory protein;
preferably, the helper protein comprises any one or a combination of two of recombinant albumin and SSB.
5. The enzyme digestion reaction solution according to claim 2, further comprising an enzyme digestion reaction buffer solution, wherein the enzyme digestion reaction buffer solution comprises metal cations, a substrate and a buffer medium;
preferably, the metal cation comprises Mg2+、K+And NH3 +Any one or a combination of more of; the substrate comprises any one or more of dNTPs, dATP and ATP; the buffer medium comprises any one or combination of more of 2-morpholinoethanesulfonic acid, acetic acid and tris.
6. The enzymatic cleavage reaction solution of claim 2, wherein the fragmentation enzyme mixture solution is composed of the recombinant topoisomerase of claim 1, the ATP-dependent nuclease, Phi29DNA polymerase, Bst II DNA polymerase, SSB, recombinant albumin, dsDNase, Tris-HCl, KCl, Tween 20, DTT, and glycerol;
preferably, the enzyme digestion reaction buffer solution is prepared from Tris-acetic acid and MgCl2KAc, dNTPs, dATP and ATP.
7. A kit comprising the recombinant topoisomerase of claim 1 or the cleavage reaction solution of any one of claims 2 to 6;
preferably, the kit further comprises a linker ligation reaction solution, wherein the linker ligation reaction solution comprises a linker ligase mixed solution and a linker ligation reaction buffer solution;
more preferably, the adaptor ligase mixture comprises a polynucleotide kinase and a ligase;
most preferably, the polynucleotide kinase is T4 polynucleotide kinase and the ligase is T4 DNA ligase.
8. Use of the recombinant topoisomerase of claim 1, the digestion reaction solution of claims 2 to 6, or the kit of claim 7 for constructing a sequencing library or for preparing a reagent for constructing a sequencing library.
9. A method of constructing a sequencing library, the method comprising:
fragmenting, end repairing and 3' tailing by using an enzyme digestion reaction solution containing the recombinant topoisomerase of claim 1, an enzyme digestion reaction solution of any one of claims 2 to 6 or an enzyme digestion reaction solution in the kit of claim 7 to obtain a fragmented product;
carrying out 5' phosphorylation and linker ligation reaction on the fragmentation product to obtain a phosphorylation and linker ligation product;
and performing PCR amplification and enrichment on the phosphorylation and adaptor connection products to obtain a sequencing library.
10. The construction method according to claim 9, wherein the reaction conditions of fragmentation, end repair and 3' tailing with A are that incubation is performed at 32 ℃ or 37 ℃ for 5-30 minutes, and then at 65 ℃ for 20-30 minutes;
preferably, the 5 'phosphorylation and linker ligation reaction is performed in the linker ligation reaction solution in the kit of claim 7, and the reaction conditions of the 5' phosphorylation and linker ligation reaction are incubation at 25 ℃ for 15-20 minutes;
more preferably, the amount of the target DNA to be added is 100pg to 1. mu.g.
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