CN112251821A

CN112251821A - Kit for quickly and efficiently constructing second-generation sequencing library

Info

Publication number: CN112251821A
Application number: CN202011124075.2A
Authority: CN
Inventors: 曾志鹏; 张鹏; 邢宽; 谢珍; 何志健; 何贵伦; 安雪茹
Original assignee: Nanjing Experimental Medicine Examines Co ltd
Current assignee: Nanjing Experimental Medicine Examines Co ltd
Priority date: 2020-10-20
Filing date: 2020-10-20
Publication date: 2021-01-22

Abstract

The invention discloses a kit for quickly and efficiently constructing a second-generation sequencing library, which comprises R1, R2 and R3, wherein R1 comprises a DNA fragmentation tail end repair tailing buffer solution, a DNA fragmentation enzyme and a tail end repair tailing enzyme, R2 comprises a connection buffer solution, a ligase and an adaptor, and R3 comprises an amplification enzyme, a USER enzyme, a universal primer and an index primer. The kit adopts nuclease with non-specific sequence, and can randomly break on DNA; adding a nonionic detergent as an enhancer in fragmentation, end repair and tailing to promote the activity of tailing enzyme, so that more end repair fragments are added with A basic groups, and the utilization rate of the fragments is improved; the U-shaped linker is used as the linker, so that the generation of linker dimer is reduced, more linkers are used for connecting DNA fragments, and the conversion rate of the library is improved.

Description

Kit for quickly and efficiently constructing second-generation sequencing library

Technical Field

The invention belongs to the technical field of high-throughput sequencing, and particularly relates to a kit for quickly and efficiently constructing a second-generation sequencing library.

Background

Second-generation sequencing, also known as next-generation sequencing (NGS), is an epoch-making revolutionary change to traditional sequencing (Sanger sequencing), which can determine sequences of hundreds of thousands to millions of DNA molecules, and makes it possible to fully analyze transcriptome or genome of a species. NGS technology can be used to sequence DNA (or RNA) of any organism, providing valuable genetic information, as a highly scalable technology, and second-generation sequencing can be applied to sequencing specific target regions or entire genomes, helping researchers to investigate and discover, and to better understand health and disease.

Before a nucleic acid sample is placed into a sequencer for sequencing, a series of treatments are usually performed on the nucleic acid sample, including nucleic acid molecule fragmentation, end repair, and the addition of specific tags and linkers at both ends of the sequence, and this process is called library construction, also called a library construction process, and NGS library construction kits usually provide a combination of reagents for completing the processes from nucleic acid fragment repair to linker addition.

At present, the process adopted for DNA library construction generally comprises the steps of sequentially carrying out end repair, purification, 3' end A addition, purification, linker connection, purification, library amplification and purification on a fragmented DNA sequence subjected to ultrasonic disruption or enzyme digestion treatment (see figure 1), wherein the whole process is long in time consumption, more purification reagents are consumed, samples are more or less lost during each purification, and the cost of the whole library construction process is high.

For trace DNA samples, high library construction transformation efficiency is one of the key parameters for improving the low-frequency variation analysis efficiency in sequencing detection. For trace DNA samples (< 50ng), it is reported by patents that the library construction transformation efficiency of currently used library construction kits of NEB, KAPAbiosystems and Illumina is low, for example, the library construction transformation efficiency of the KAPA hyper library construction kit of KAPAbiosystems for 25ng of DNA fragments of 170bp is 2.21% -8.84%, and the library construction transformation efficiency for 50ng of DNA fragments of 170bp is 4.43% -8.84%.

Therefore, there is still a need for improvement of the prior art to overcome the disadvantages of long process, high cost and low conversion rate of database construction.

Disclosure of Invention

The invention aims to provide a kit for quickly and efficiently constructing a second-generation sequencing library, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a kit for constructing a second-generation sequencing library rapidly and efficiently comprises R1, R2 and R3;

the R1 comprises a DNA fragmentation end repair tailing buffer, a DNA fragmentation enzyme and an end repair tailing enzyme;

the R2 comprises a connection buffer, a ligase and a linker;

the R3 comprises an amplification enzyme, a USER enzyme, a universal primer and an index primer.

Preferably, the DNA fragmentation end repair tailing buffer comprises 10-50mM Tris-HCl, 10-50mM MgCl₂1-10mM DTT, 20-100 MuM dNTP, 0.2-1.0mM dATP, 20-80mM NaCl, 0.1-1mg/ml BSA, 0.1-0.5% TritonX-100 by volume ratio, and 0.1-1% Enhance by volume ratio.

In any of the above schemes, preferably, the Enhance may be one or a combination of two of tween 20 and NP 40.

Preferably in any one of the above schemes, the DNA fragmenting enzyme is a non-specific nuclease which is vibrio halophilus vvn (vibrio vulnifica) nuclease and T7 endonucleoclean I.

In any of the above embodiments, preferably, the end-repair tailing enzyme is a combination of T4 polynucleotide kinase, T4 DNA polymerase, Taq DNA polymerase, and Klenow enzyme.

In any of the above schemes, preferably, the linker is a U-shaped linker, and the sequence of the linker is shown in SEQ ID No. 1:

SEQ ID NO.1：5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；

wherein, the first G base is subjected to phosphorylation modification, the connecting bond between the last two nucleotides at the 3' end is subjected to thiophosphorylation modification, and the U base is dUTP.

The invention has the technical effects and advantages that: 1. the DNA fragmentation enzyme uses Vvn nuclease, which is a nuclease with non-specific sequence, can randomly generate nicks on DNA and has no base preference;

2. the Enhance is added in DNA fragmentation, end repair and A tail addition, so that the tail addition activity of taq enzyme can be promoted, more end repair fragments are added with A basic groups, more A fragments are connected with a linker, and the utilization rate of the fragments is improved;

3. the U-shaped linker is used as the linker, so that the generation of linker dimer is reduced, more linkers are used for connecting DNA fragments, and the conversion rate of the library is improved.

Drawings

FIG. 1 is a flow of conventional second generation sequencing library construction according to the present invention;

FIG. 2 is a second generation sequencing library building process using the kit of the present invention;

FIG. 3 is a diagram of a library 2100 constructed in test example 1;

FIG. 4 is a diagram of a library 2100 constructed in test example 2.

Detailed Description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Some of the kits mentioned in the background art have low conversion rates in the case of library construction, especially in the case of micro samples, which results in a large loss of sample. For micro samples, methods of fragmentation with enzyme sections are suggested to reduce sample loss.

The fragmenting enzymes which can be used for nucleic acid disruption are reported on the market, and the fragmenting enzymes which are used for high-throughput sequencing comprise DNase I, Endonuclease V, Tn5 transposase, restriction Endonuclease and the like.

DNase I can fragment dsDNA in the presence of different ions, such as any site of double-stranded DNA randomly sheared by DNase I in the presence of magnesium ions; in the presence of divalent manganese ions, DNaseI can cut a DNA double strand at the same site to form a flat end or a sticky end with 1-2 nucleotides protruding. However, the actual operation is susceptible to various conditions such as the amount of enzyme used, the reaction temperature, the purity of substrate DNA, etc. In addition, DNase I is found to have preference to sites beside pyrimidine nucleotides, and the diversity of the final library is greatly influenced. Endonuclease V generally recognizes specific sites, and random introduction of uracil can be used for random fragmentation of DNA. Due to the difference of the base sequence composition, the GC content of the sample has potential influence on the Endonuclease V fragmentation effect, and the practical operation, especially the application of NGS, is limited by a plurality of conditions. Tn5 transposase also cleaves DNA, but its fragmentation preference is often subject to industry challenges. Tn5 was statistically significantly biased around 9 bases on either side of the insertion site. There are also companies that use a combination of several restriction enzymes, and although increasing the number of restriction enzymes can alleviate the preference to some extent, there is always a preference for sequences for restriction enzymes.

The invention breaks DNA by using the combination of Vvn nuclease and T7 Endonuclease I which are sequence non-specific nuclease. Vvn nuclease can randomly generate nicks on DNA, and T7 endonucleolease I recognizes this nick and cuts on the complementary strand, thereby generating a DNA double strand break. The DNA can be cut into DNA fragments with specific sizes only by changing the cutting time, and the method is independent of the initial Input DNA amount and the Input DNA length.

The DNA used by the DNA fragmenting enzyme used in the present invention may be various prokaryotic eukaryotic microorganism genomes, animal and plant genomes, human and mouse genomes, etc., or cDNA, but it is necessary to ensure that the DNA is soluble in a divalent cation-free solvent and insoluble in a divalent cation-free solvent, and if soluble in a divalent cation-free solvent, it is necessary to purify it beforehand. Since divalent cations inhibit the activity of the fragmenting enzyme.

In order to further improve the utilization rate of the fragments, the non-ionic detergent is added as an enhancer of the taq enzyme during tailing to promote the activity of the taq enzyme, so that more terminal repair fragments are added with A basic groups, more A-added fragments are connected with a linker, and the utilization rate of the fragments is promoted.

In order to further increase the conversion rate of the library, the present invention uses a U-shaped linker in the linker ligation step. This type of linker, which is blocked at one end, reduces the inter-linker reactions and thus reduces the production of linker dimers, allowing more linkers to be efficiently used and thus more available fragments of the linker to be sequenced.

The first embodiment is as follows:

a kit for constructing a second-generation sequencing library rapidly and efficiently comprises R1, R2 and R3;

the R2 comprises a connection buffer, a ligase and a linker;

The DNA fragmentation end repair tailing buffer comprises 10mM Tris-HCl and 10mM MgCl₂1mM DTT, 20. mu.M dNTP, 0.2mM dATP, 20mM NaCl, 0.1mg/ml BSA, 0.1% TritonX-100 by volume, and 0.1% Twen20 by volume.

The DNA fragmenting enzyme is nonspecific nuclease and T7 Endonuclease I, wherein the nonspecific nuclease is Vvn nuclease.

The tail end repair tailing enzyme is a combination of T4 polynucleotide kinase, T4 DNA polymerase, Taq DNA polymerase and Klenow enzyme.

The joint is a U-shaped joint, and the sequence of the joint is shown in SEQ ID NO. 1:

5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；

Example two:

the R2 comprises a connection buffer, a ligase and a linker;

The DNA fragmentation end repair tailing buffer comprises 30mM Tris-HCl and 30mM MgCl₂5mM DTT, 130. mu.M dNTP, 0.6mM dATP, 50mM NaCl, 0.5mg/ml BSA, 0.3% by volume TritonX-100, 0.5% by volume Twen 20.

The DNA fragmenting enzymes are Vvn nuclease and T7 Endonuclease I.

5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；

Example three:

the R2 comprises a connection buffer, a ligase and a linker;

The DNA fragmentation end repair tailing buffer comprises 50mM Tris-HCl and 50mM MgCl₂10mM DTT, 100. mu.M dNTP, 1.0mM dATP, 80mM NaCl, 1mg/ml BSA, 0.5% by volume Triton X-100, 1% by volume Twen 20.

The DNA fragmenting enzymes are Vvn nuclease and T7 Endonuclease I.

5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；

Example four:

the R2 comprises a connection buffer, a ligase and a linker;

The DNA fragmentation end repair tailing buffer comprises 30mM Tris-HCl and 30mM MgCl₂5mM DTT, 130. mu.M dNTP, 0.6mM dATP, 50mM NaCl, 0.5mg/ml BSA, 0.3% Triton X-100 by volume, 0.5% Twen20 by volume, 0.5% NP4 by volume0。

The DNA fragmenting enzymes are Vvn nuclease and T7 Endonuclease I.

5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；

A method for quickly and efficiently constructing a second-generation sequencing library comprises the following steps in sequence:

s1, adding DNA fragmentation tail end repairing tailing buffer solution, DNA fragmentation enzyme and tail end repairing tailing enzyme into a test tube containing DNA, fully mixing, incubating at 4 ℃ for 1 minute, 32 ℃ for 5-35 minutes, and 65-75 ℃ for 15-30 minutes to obtain a DNA fragment with a repaired tail end and an added tail A;

s2, adding a linker, a connection buffer solution and a ligase into the DNA fragments, fully mixing, incubating for 15-30 minutes at 20-37 ℃ to obtain fragments with the linker, carrying out PCR enrichment on the fragments with the linker, and then purifying by a magnetic bead purification step;

s3, adding high fidelity amplification enzyme, universal primer and index primer into the fragments with joints, and amplifying the fragments with joints to obtain a DNA library.

In order to better understand the use of the present invention, two specific test examples will be shown below.

Test example 1: human genome DNA is used as an experimental material to construct a rapid and efficient sequencing library.

The library building process is shown in fig. 2, and the specific experimental steps are as follows:

(1) sample preparation: the concentration of the genomic DNA was precisely determined by the Qubit, and 10ng of the volume of the fragment to be added was calculated from the determined concentration, and the remainder was made up to 27.6. mu.L with water (NFW);

(2) fragmentation, end repair and addition of a: the specific reaction system is shown in table 1:

TABLE 1

Components	Volume (μ L)
		DNA	27.6
DNA fragmentation end repair tailing buffer	10
		DNA fragmenting enzyme	10
End repair tailgating enzyme	2.4
		Total of	50

Wherein, the tail end repairing tailing enzyme comprises the following components: t4 polynucleotide kinase 0.2U/. mu.L, T4 DNA polymerase 0.02U/. mu.L, Klenow fragment 0.02U/. mu.L, Taq enzyme 0.02U/. mu.L;

the DNA fragmentation end repair tailing buffer comprises the following components: Tris-HCl 50mM, MgCl₂10mM, 1mM DTT, 20 μ M dNTP, 0.2mM dATP, 20mM NaCl, 0.1mg/ml BSA, 0.1% TritonX-100 by volume and 0.5% Twen20 by volume;

after the system is prepared, placing the reaction tube in a PCR instrument or other thermal incubation reaction instruments, and incubating for 22 minutes at 32 ℃; incubating for 30 minutes at 65 ℃, and taking out the reaction tube for the next reaction;

(3) connecting a joint: adding a connecting reagent into the repair fragments treated in the step (2) and the reaction solution to connect the adaptor and the DNA fragments, wherein the reaction system is shown in the following table 2:

TABLE 2

Components	Volume (μ L)
		The product of the last step	50
Joint	8
		Ligation buffer	16
Ligase	6
		Total of	80

After the system is prepared, placing the reaction tube in a PCR instrument or other thermal incubation reaction instruments, setting the temperature at 25 ℃ for 15min, and taking out the reaction tube after the reaction is finished to carry out the next purification process;

the sequence of the linker used in the step is shown as SEQ ID NO.1, and the linker is formed by annealing and cyclization of the SEQ ID NO. 1;

(4) purification of the fragment with the linker: purifying the reaction system after connection by using 0.8X magnetic beads; then respectively rinsing with 80% ethanol and eluting with NFW to obtain purified fragments with connectors;

(5) and (3) PCR amplification: PCR enrichment is carried out on the purified band-joint fragments by using the amplilase, the USER enzyme, the universal primer and the index primer to obtain a DNA library, which is shown in the table 3:

TABLE 3

Components	Volume (μ L)
		The product of the last step	17
High-fidelity amplification enzyme mixed solution	25
		USER enzyme	3
General primer (10. mu.M)	2.5
		index primer (10. mu.M)	2.5
Total of	50

Then, PCR amplification was performed according to the PCR program of table 4;

TABLE 4

(6) And (3) post-PCR purification: after the reaction is finished, taking out the PCR tube, performing instantaneous centrifugation, collecting all liquid to the bottom of the tube, purifying by using magnetic beads with the same volume, and eluting by using 20 mu l of nuclease-free water after the purification is finished, so as to obtain the PCR tube;

(7) performing quality inspection on the library: use of

The concentration of the library is detected by the dsDNA HS Assay Kit, the size of the fragment of the library is analyzed by using a 2100 bioanalyzer, the operation is carried out according to the specification requirements, and the quality inspection shows that the method is rapid and the total amount of the constructed library is high as shown in Table 5 and figure 3.

TABLE 5

Initial amount ng	Concentration ng/. mu.l	Volume μ l	Total amount ng
				10	20.2	20	404

Test example 2:

in contrast to the first example, the DNA fragmentation end-repair tailing buffer composition was: Tris-HCl 40mM, MgCl₂ 5mM，DTT 3mM，dNTP 30μM，dATP 0.3mM，NaCl 30mM，BSA 0.2mg/ml，Tr0.15% of itonX-100 by volume and 0.75% of Twen20 by volume;

the tail end repairing tailing enzyme comprises the following components: t4 polynucleotide kinase 0.1/. mu.L, T4 DNA polymerase 0.02U/. mu.L, Klenow fragment 0.01/. mu.L, Taq enzyme 0.02U/. mu.L;

the rest is the same as the first embodiment, the library construction effect is basically the same as the first embodiment 1, and the results are shown in the following table 6 and fig. 4.

TABLE 6

Initial amount ng	Concentration ng/. mu.l	Volume μ l	Total amount ng
				10	19.8	20	396

Therefore, the buffer solution system and the enzyme system are improved, the shape of the joint is changed, the micro-initial library construction is rapid and efficient, the method effectively solves the problems that the total amount of part of precious and difficultly obtained samples is too low, the library construction can not be carried out for sequencing, or the library construction conversion rate is low, and the library quality is poor, improves the application range of the second-generation sequencing, and has wider application range.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A kit for quickly and efficiently constructing a second-generation sequencing library is characterized in that: including R1, R2, and R3;

the R2 comprises a connection buffer, a ligase and a linker;

2. The kit for rapidly and efficiently constructing the second-generation sequencing library according to claim 1, wherein: the DNA fragmentation end repair tailing buffer comprises 10-50mM Tris-HCl and 10-50mM MgCl₂1-10mM DTT, 20-100 MuM dNTP, 0.2-1.0mM dATP, 20-80mM NaCl, 0.1-1mg/ml BSA, 0.1-0.5% TritonX-100 by volume ratio, and 0.1-1% Enhance by volume ratio.

3. The kit for rapidly and efficiently constructing the second-generation sequencing library according to claim 1, wherein: the Enhance is one or the combination of two of Twen20 and NP 40.

4. The kit for rapidly and efficiently constructing the second-generation sequencing library according to claim 1, wherein: the DNA fragmentation enzyme is nonspecific nuclease and T7 Endonuclease I, wherein the nonspecific nuclease is Vvn nuclease.

5. The kit for rapidly and efficiently constructing the second-generation sequencing library according to claim 1, wherein: the tail end repair tailing enzyme is a combination of T4 polynucleotide kinase, T4 DNA polymerase, Taq DNA polymerase and Klenow enzyme.

6. The kit for rapidly and efficiently constructing the second-generation sequencing library according to claim 1, wherein: the joint is a U-shaped joint, and the sequence of the joint is shown in SEQ ID NO. 1: SEQ ID NO. 1: 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', respectively;