CN112391442A - Nucleic acid sample processing method, sequencing method and kit - Google Patents

Abstract

The invention provides a DNA sample processing method, a sequencing method and a kit. The method for processing the DNA sample comprises the steps of fragmenting, repairing the tail end and adding dA tail to the DNA in a first pre-mixing system to obtain a first product; the first pre-mix system is said to comprise pre-mixed enzymes, which are said to comprise DNA-fragmenting enzymes, polynucleotide kinases and DNA polymerases. Based on a test and an optimized mixed enzyme system, a plurality of enzymes are mixed in the same reaction system, DNA breakage, terminal repair and dA tail addition are realized in one step, the method is simple, convenient and quick, the operation time of pre-machine sample treatment is saved, and the industrialization is facilitated.

Description

Nucleic acid sample processing method, sequencing method and kit

Technical Field

The invention belongs to the field of biomacromolecule detection and analysis, and particularly relates to a nucleic acid sample processing method, a sequencing method and a kit.

Background

High throughput sequencing, including secondary sequencing and single molecule sequencing, involves the processing of nucleic acid samples prior to on-machine detection. In particular, nucleic acid sequencing based on chip detection requires the processing of the nucleic acid to be detected (template) to attach it to the chip for sequencing.

Currently available chip-based detection sequencing platforms are generally sold as a kit with a specific sequence (probe) on the kit, and the template generally needs to be processed by various methods to contain the specific sequence or at least a part of the sequence matching the specific sequence so that the template can be bound to the chip; the various treatments referred to generally involve, for long nucleic acid fragments, such as genomic DNA, in particular, mechanical or enzymatic cleavage of nucleic acids to obtain nucleic acid fragments, repair of the nucleic acid fragments such as filling in their ends and/or making them have specific cohesive ends so that the nucleic acid fragments can be ligated with specific sequences (linkers) to obtain a sequencing library.

At present, there are various commercially available kits for each step of the above-mentioned pre-processing of nucleic acids, for example, a fragmentation kit, an end-repair optional dA-tailed kit, a linker ligation kit, and a kit for each step of purification, including a kit for detecting and/or removing a specific reagent interfering with a subsequent reaction in a previous reaction system, and the like. Generally, no commercially available kit discloses a specific formulation thereof, including the kind, characteristics, concentration, specific reaction solution configuration, etc. of the enzyme.

The rapid and simple treatment method of pre-nucleic acid, including the related solution system, is in need of further development and improvement.

Disclosure of Invention

The invention provides a method for processing a DNA sample, which mixes a plurality of enzymes in the same reaction system to realize DNA breakage, end repair and dA tail addition in one step, is simple, convenient and quick, saves the operation time of sample processing before loading and is beneficial to industrialization; the invention also provides a sample sequencing method, which comprises the steps of capturing the DNA sample processed by the DNA sample processing method provided by the invention through a probe, and sequencing by the second generation technology and the third generation technology; in addition, the invention provides a kit, wherein the End Prep Mix in the kit is formed by mixing a plurality of enzymes and a buffer solution, and the kit can be used for processing a DNA sample and sequencing the sample, can simplify the operation, and is simple, convenient and quick.

The invention provides a method for processing a DNA sample, which comprises the steps of fragmenting, repairing and adding dA tail to the DNA in a first premixing system to obtain a first product; the first pre-mix system includes a pre-mixed enzyme that includes a DNA-fragmenting enzyme, a polynucleotide kinase, and a DNA polymerase.

It will be appreciated that the action of the DNA fragmenting enzyme in the above method fragments the DNA sample to the size of the desired fragments, and in embodiments of the invention, the DNA fragmenting enzyme may be selected from T7 DNO I, Pediococcus pestis phage phiA1122 endonuclease, phage PhiYe03-12 endonuclease, phage T3 endonuclease, phage T3 DNO endonuclease, Pseudomonas phage gh-1 endonuclease, Pseudomonas putida KT2440 DNO I and Rose phage S101RP endonuclease I, and the like.

In one example, the DNA in the above method is not less than 5Kb, and the first product after fragmentation is 100-400 bp. Based on the restriction of the sequencing length of the sequencing instrument, in one example, the size of the selected first product is 100-300 bp.

In one example, the amount of DNA in the above method is 10ng to 60 ng; preferably, the content of the DNA in the method is 15ng-50ng, the content of the DNA is directly related to the size of a reaction system, the amount of enzyme used and the amount of a required library, the amount of the library constructed with too small amount of the DNA is too small, sequencing obtained data cannot meet the requirement, and meanwhile, if the content of the DNA is too large, the DNA cannot be sufficiently fragmented and subjected to a joint adding reaction in a certain system, and a lot of invalid fragments appear in the constructed library. The DNA content is 15ng-50ng, and after multiple tests, the reaction can be effectively carried out at the fragmentation reaction temperature of 32 ℃, and meanwhile, the library constructed by utilizing the DNA content meets the requirement of on-machine sequencing.

In one example, the first pre-mix system further comprises Mg²⁺Said Mg²⁺Preferably, the concentration of Mg in the first pre-mixture is 60 mM-100 mM²⁺The concentration of (B) is 60 mM-75 mM. Different enzymes for Mg²⁺In one embodiment of the invention, the volume ratio of the first pre-mixed system to the total system of genomic DNA fragmentation, end repair and dA-tailed addition reactions is 1: 5, namely in the total reaction system, mixing Mg selected for enzyme reaction²⁺The concentration of (A) is 12 mM-15 mM, and Mg is selected²⁺The concentration of (A) is 6mM higher than that selected for the conventional enzyme reaction.

In one example, in the first pre-mixing system of the above method, the DNA-fragmenting enzyme is present in an amount of at least 2 times the polynucleotide kinase in total activity units U, and the DNA polymerase is selected from at least one of DNA polymerase I and Taq DNA polymerase, and preferably, the DNA polymerase consists of Klenow fragment of DNA polymerase I and Taq DNA polymerase. The polynucleotide kinase is T4 polynucleotide kinase.

In one example, the amounts of the DNA fragmenting enzyme, the polynucleotide kinase, the Klenow fragment of DNA polymerase I and the Taq DNA polymerase in the first pre-mix system are in a ratio of [25,35]: 6,10]: 1, 3: 3,5], the total enzyme activity units U, which is optimized by multiple experiments and can simultaneously satisfy the requirements of the sample for different enzymes. In one embodiment of the invention, the total enzyme activity unit ratio of the DNA-fragmenting enzyme, the polynucleotide kinase, the Klenow fragment of DNA polymerase I, and the Taq DNA polymerase is 18: 6: 1:1, the concentration of the taq enzyme in the first pre-mixing system is (1/3-2/3) U/ul. Wherein Taq enzyme activity unit is defined as the amount of enzyme required to incorporate 10nmol of dNTP into the acid-insoluble precipitate within 30 minutes at 37 ℃ and is defined as 1 activity unit (U); the Klenow fragment enzyme activity unit is defined as the amount of enzyme required to incorporate 10nmol of dNTP into the acid-insoluble precipitate within 30 minutes at 37 ℃ and is defined as 1 activity unit (U); nucleotide kinase activity unit is defined as the amount of enzyme required to incorporate 1nmol of [ gamma-32P ] ATP into the acid-insoluble precipitate within 30 minutes at 37 ℃ as 1 activity unit (U); the unit of activity of DNA-fragmenting enzyme is defined as the amount of enzyme required to convert 1. mu.g of supercoiled pUC (AT) into a linear form (double strand cleavage) or a nicked form (single strand cleavage) in a reaction of 20ml at 37 ℃ in 30 minutes as 1 unit of activity (U).

Any one of the DNA sample processing methods is based on a test and an optimized mixed enzyme system, so that a plurality of sample processing modules are efficiently realized in one step in the same reaction system, including DNA breakage, terminal repair and dA tail addition, the obtained processed nucleic acid is suitable for being placed on various sequencing platforms for detection, the operation is simple, convenient and quick, the time required by sample processing before loading is saved, and the industrialization is facilitated; further, the product obtained through the treatment can be directly subjected to the next treatment process without being separated or purified from the reaction system.

In one example, the above method further comprises providing at least one end of the first product with a predetermined sequence in a second pre-mix system to obtain a second product, the second pre-mix system comprising the first pre-mix system and the predetermined sequence.

Further, the second pre-mix system may further comprise a ligase, such as T4 DNA ligase.

Further, the predetermined sequence is a double-stranded DNA, and is composed of a first strand and a second strand that are complementary, and the predetermined sequence has at least one single-stranded end.

In one example, the 5' end of the first strand has a phosphate group, the 3' end of the second strand does not have a hydroxyl group, the 5' end of the first strand is linked to the first product, the ligation reaction is performed by using the predetermined sequence, only the first strand is linked to the first product, and the library constructed by the method can be directly used for sequence capture in the library construction process without PCR.

Further, 80 ℃ or more (Tm1-Tm2) or more is 10 ℃ or more and 90 ℃ or more Tm1 or more is 50 ℃ or more, Tm1 is the dissolution temperature of the first chain, and Tm2 is the dissolution temperature of the second chain. Specifically, Tm1 is 71 ℃, Tm2 is 45.6 ℃, and the difference of the two strand dissolution temperatures is used for reducing the influence of the second strand on the capture efficiency in the sequence capture process.

In one example, the predetermined sequence is selected from SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO:1 and SEQ ID NO:3 and/or SEQ ID NO:4 and SEQ ID NO: 5.

any of the above methods, based on the method of any one of 1 to 12, can make the nucleic acid to be tested have a predetermined sequence (also referred to herein as a linker) by ligation and/or polymerization, and can rapidly and efficiently prepare a nucleic acid to be tested (sequencing library) containing a specific sequence by using the reaction system and the designed linker involved in any one of the methods, so as to meet the requirements of the subsequent computer reaction.

In a second aspect, the present invention provides a method for determining a DNA sequence, comprising:

processing the DNA by using the method for processing the DNA sample to obtain a first product and a second product;

the second product is attached to the surface of the solid substrate and the second product is sequenced to determine the sequence of at least a portion of the DNA.

In one example, the second product carries an optically detectable label, such as a fluorescent molecule.

In one example, the solid substrate has a probe on its surface, and the second product is attached to the solid substrate surface via the probe, wherein the probe has a length of 20-80 nt. The solid matrix is selected from at least one of glass, plastic and magnetic beads.

In one example, the predetermined sequence is double-stranded DNA consisting of a first strand and a second strand that are complementary, and the first strand of the predetermined sequence has a single-stranded end. Further, a first strand is fully complementary to the probe, the first strand being no longer than the probe. Further, Tm2-5 ℃ and T are more than or equal to Tm1-5 ℃, T is the reaction temperature for connecting the second product to the surface of the solid phase substrate, the temperature for performing sequence capture by the probe is T, Tm1 is the dissolution temperature of the first chain, and Tm2 is the dissolution temperature of the second chain, and the reaction temperature can be used for effectively reducing the influence of the second chain on the sequence capture of the probe.

In one example, the method for determining a DNA sequence further comprises denaturing the second product before attaching the second product to the surface of the solid phase matrix.

In the method for determining the DNA sequence, the probe and/or the reaction condition meeting the specific requirements are designed and selected, so that the sequencing library can be efficiently connected to the surface of the solid-phase matrix and stably connected, and the template information can be obtained by stably and efficiently sequencing; meanwhile, any of these methods has the advantages and technical features of any of the foregoing exemplary methods, and will not be described herein again.

In a third aspect, the present invention provides a kit comprising a first pre-mixing system for performing the method for treating a DNA sample and the method for determining a DNA sequence as described above. When the kit containing a premixing system is used for treating the DNA of a sample, DNA breaking, end repairing and dA tail adding can be realized in one step, the method is simple, convenient and quick, and the DNA treatment time is saved.

In one example, the kit further comprises the second pre-mix system.

In one example, the kit further comprises a probe for performing the above-described DNA sequence determination method, the second product being attached to the surface of the solid substrate via the probe, the probe having a length of 20 to 80nt of DNA sequence.

Further, the probe is complementary to the first strand, the probe is not shorter than the first strand, the predetermined sequence is a double-stranded DNA composed of the complementary first strand and second strand, the predetermined sequence has a single-stranded end, and the single-stranded end is located on the first strand.

The kit of any of these, which can be used to carry out any of the methods described above, provides any of the aforementioned advantages to any of the methods, and is industrially advantageous. Any kit can be used for pre-processing nucleic acid; alternatively, and for nucleic acid sequence determination.

Drawings

FIG. 1: detection of labchip, a product after linker addition, in example 2, from F4, F5, F6 and F74 samples;

FIG. 2: labchip detection maps of F4, F5, F6, and F74 sample PCR products in example 2;

FIG. 3: detection maps of the product labchip of N5, N6, N13, N14, N21, N22, N23, N24, O5, O6, O11 and O1212 samples after disruption, end-repair and A addition in example 3.

Detailed Description

Unless otherwise defined, the terms used herein have the ordinary meaning in the art to which this invention belongs.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

Description of materials and reagents

Linker 1 (D9-T): a double-stranded nucleic acid formed by two single-stranded nucleic acids (a first strand and a second strand), that is, a double-stranded nucleic acid formed by SEQ ID NO:1 and SEQ ID NO:2, or a double-stranded nucleic acid formed by SEQ ID NO:1 and SEQ ID NO: 3:

first strand 5'-AGATGTGTATAAGAGACAGT-3' (SEQ ID NO:1)

Second strand 5'-ACTGTCTCTTATACACATCTGAGTGGAACTGGATGGTCGCAGGTATCAAGGATT-3' (SEQ ID NO:2) or 5'-CTGTCTCTTATACACATCTGAGTGGAACTGGATGGTCGCAGGTATCAAGGA-3' (SEQ ID NO:3)

And (3) joint 2: a double-stranded nucleic acid formed from two single-stranded nucleic acids (a first strand and a second strand), e.g., a P5 and/or P7 linker that matches part of the ILLUMINA sequencing platform; commercially available kits, such as nunoprazan:

the first chain: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3' (SEQ ID NO:4)

The second chain: 5'-p-GATCGGAAGAGCACACGTCTGAACTCCAGTC-3' (SEQ ID NO:5)

(-s-for thio, -p for phosphorylating)

The examples, where specific techniques or conditions are not indicated, are in accordance with routine experimentation or with the manufacturer's instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

The following illustrates the method of making the kit.

The kit comprises a tube 1, wherein the tube 1 is a mixed system with a terminal repair function (also called End Prep Mix). In one instance, tube 1 is a solution comprising a polynucleotide kinase and a plurality of DNA polymerases. In another case, tube 1 is a solution comprising a DNA-fragmenting enzyme, a polynucleotide kinase, and a plurality of DNA polymerases.

The DNA polymerase can fill in the ends of the fragmented nucleic acid fragments, and preferably, the selected DNA polymerase has an amplification function and a repair and proofreading function, and is selected from one or two of Taq DNA polymerase (abbreviated as Taq enzyme) and Klenow fragment; the Polynucleotide Kinase can be selected from T4 Polynucleotide Kinase (T4 Polynucleotide Kinase).

In one case, the DNA polymerase is a mixture of Taq enzyme and Klenow fragment, the enzyme activity unit (U) ratio of Taq enzyme, Klenow fragment and polynucleotide kinase in tube 1 is 1:1:6, and the concentration of Taq enzyme in tube 1 is (1-2) U/ul. Here, the unit of activity of each enzyme is defined as follows: taq enzyme activity unit: the amount of enzyme required to incorporate 10nmol of dNTP into the acid-insoluble precipitate at 37 ℃ for 30 minutes is defined as 1 activity unit (U); klenow fragment activity units: the amount of enzyme required to incorporate 10nmol of dNTP into the acid-insoluble precipitate at 37 ℃ for 30 minutes is defined as 1 activity unit (U); nucleotide kinase activity units: the amount of enzyme required to incorporate 1nmol of [ gamma-32P ] ATP into the acid-insoluble precipitate at 37 ℃ for 30 minutes was defined as 1 activity unit (U).

In one case, the tube 1 further contains a DNA-fragmenting enzyme, and the tube 1 is produced by preparing a mixed enzyme solution a other than the DNA-fragmenting enzyme and then adding the DNA-fragmenting enzyme solution to the mixed enzyme solution a, for example, a DNA-fragmenting enzyme solution and the mixed enzyme solution a in a volume ratio of 1: 2 mixing such that the DNA fragmentation enzyme, the mixture of Klenow fragments, the enzyme activity unit (U) ratio of Taq enzyme, Klenow fragment and polynucleotide kinase in tube 1 is 18: 1:1:6, wherein the concentration of the taq enzyme in the tube 1 is 2/3 of the concentration of the taq enzyme in the mixed enzyme solution A; the DNA fragmenting enzyme may be selected from the group consisting of the Tiangen DNA fragmenting kit NG305-02, the Qiagen CM0162 fragmenting enzyme kit or the NEB fragmenting enzyme. The Taq enzyme, Klenow and polynucleotide kinase contained in the enzyme mixture A may be selected from those sold by NEB, Tiangen, Novozam (Vazyme), and the like. DNA fragmenting enzyme Activity Unit 1. mu.g of supercoiled pUC (AT) was converted into a linear form (double strand cleavage) or a nicked form (single strand cleavage) in a reaction of 20ml at 37 ℃ within 30 minutes.

When preparing the tube 1, preparing or purchasing each individual enzyme solution separately, and mixing each individual enzyme solution; it is also possible to prepare a mixed enzyme solution and a buffer solution separately and mix them, for example, by mixing them in a volume ratio of 1:1 to prepare a tube 1,.

In one case, in preparing tube 1, separate enzyme solutions including separate DNA fragmentation enzymes, T4 polynucleotide kinase, Taq DNA polymerase and Klenow fragment kit were selected for purchase from each merchant; the individual enzyme solutions in each commercially available kit were then mixed in the specified ratio and MgCl was additionally added₂Solutions, e.g. by additionally adding MgCl of not less than 60mM₂Thereby producing a tube 1.

In one case, the various enzymes in tube 1 are placed together in the following solution system: the 10 × Buffer comprises 700mM Tris-HCl pH 7.6,120-150mM MgCl2,50mM DTT and 1mM dNTPs; here, 1mM dNTPs means that the concentration of each dNTP is 1 mM. The ratio of the total volume of the various enzymes in tube 1 to the volume of 10x Buffer was 1:1, the mixing system of the tube 1 obtained at this time is 5x End Prep Mix, that is, when the reaction of DNA fragmentation, End repair and dA tail addition is performed by using the mixing system of the tube 1, the volume ratio of the mixing system of the tube 1 to the total reaction system is 1: 5; the specific components and concentrations of Buffer in tube 1 are respectively: 350mM Tris-HCl pH 7.6,60-75mM MgCl2,25mM DTT and 0.5mM dNTPs, and the concentration of taq enzyme in tube 1 is (1/3-2/3) U/ul.

In some cases, the kit further comprises a tube 2, the tube 2 being a mixed system with Ligation function (Ligation Mix) consisting of DNA ligase and a ligase buffer system. The DNA Ligase may be Blunt/TA Ligase, e.g., T4 DNA Ligase. In some cases, the kit further comprises a tube 2, the tube 2 being a mixed system with Ligation function (Ligation Mix) consisting of DNA ligase and a ligase buffer system. The DNA Ligase may be Blunt/TA Ligase, e.g., T4 DNA Ligase. The concentration of each substance of the 2x Ligation Mix is: t4 DNA Ligase buffer: 80-100mM Tris-HCl pH 7.8, 20mM MgCl2,20mM DTT, 10mM ATP, 50ug/ml BSA, and T4 DNA Ligase activity unit concentration of 60U-100U/ul. Different concentrations of Ligation Mix can be configured as required. The unit of activity of T4 DNA Ligase is defined as the amount of enzyme required to ligate 50% of 6ug of lambda DNA HindIII digest in 20ul reaction 50mM Tris-HCl pH 7.5, 10mM MgCl2,10mM DTT, 5mM ATP, 25ug/ml BSA at 16 ℃ for 30 min. The Ligation Mix may be selected from the reagents in the NEB kit under the trade name M0367S.

In some cases, the kit further comprises tube 3, tube 4, and tube 5, etc., e.g., tube 3 comprising PCR reaction reagents, tube 4 comprising a linker, and tube 5 comprising a primer, etc. For example, linker 1(D9-T) comprising an independent packaging useful for the construction of a third generation sequencing library, linker 2 comprising an independent packaging suitable for the construction of a second generation sequencing library, e.g., the ILLUMINA platform; the PCR amplification reagent can be selected from a common PCR amplification kit (containing taq enzyme) or a high-fidelity PCR amplification kit (containing high-fidelity enzyme, such as pfu), and can be selected from PCR amplification kits of companies such as Sammy, Moke, Beckman and the like.

By using the kit, libraries suitable for various sequencing platforms can be efficiently constructed, including but not limited to the second generation sequencing platform and the third generation sequencing platform.

Example 2

This example provides the use of the kit of example 1 in library construction. In this example, 4 parallel experiments were performed, wherein the samples used were F4, F5, F6, and F7 genomes, and the genome DNA was subjected to preliminary evaluation of genome quality before fragmentation, to ensure that the genome was present in a solvent free of metal ion chelators or other salts, and to detect genome integrity by gel electrophoresis. The specific steps of library construction are as follows:

1. genomic DNA fragmentation, end repair and dA tailing

This step fragments the DNA while filling in the ends of the fragmented DNA, phosphorylates it at the 5 'end and adds a dA tail at the 3' end.

The reaction system was configured in a PCR tube as shown in table 1:

TABLE 1

H2O	14ul
		End Prep Mix	4ul
DNA(25ng/ul)	2ul
		Total	20ul

The PCR tube was placed in a PCR apparatus, and the reaction conditions were set as shown in Table 2:

TABLE 2

Temp	Time
		105 deg.C thermal cover	On
32℃	15-20min
		65℃	10min
4℃	Hold

And immediately entering the next operation after the reaction is finished.

2. Joint connection

After the reaction of step 1, a linker ligation system was prepared in a PCR tube as shown in Table 3:

TABLE 3

Components	Volume of
		Product of step 1	20ul
Joint 1 (concentration 15uM)	5ul
		Ligation Mix(NEB M0367S)	25ul
Total	50ul

Reaction conditions are as follows: standing at room temperature for 15 min.

3. Purification of

The reaction product was purified using VAHTS DNA Clean Beads, the purification procedure is as follows:

1) the adaptor-ligated system was transferred to a 1.5mL EP tube, 0.8X (40. mu.L) magnetic beads were added, and the mixture was pipetted and mixed 10 times, and allowed to stand at room temperature for 3 min.

2) Place 1.5mL of EP tube on magnetic stand, let stand for 2-3min, and remove the supernatant.

3) Freshly prepared 200. mu.L of 80% ethanol was used for bead wash on the shelf.

4) After removal of the ethanol, it was centrifuged instantaneously, placed on a magnetic stand and removed with a 10 μ L gun.

5) And opening the cover to dry the magnetic beads for about 5-10min until the ethanol is completely volatilized.

6) Add 22. mu.L ddH₂Eluting with O, mixing, standing at room temperature for 3min, placing on magnetic frame for 3min, recovering 20 μ L product, adding 1.2x (24ul) magnetic bead, blowing and mixing for 10 times, and standing at room temperature for 3 min.

7) Place 1.5mL of EP tube on magnetic stand, let stand for 2-3min, and remove the supernatant.

8) Freshly prepared 200. mu.L of 80% ethanol was used for bead wash on the shelf.

9) After removal of the ethanol, it was centrifuged instantaneously, placed on a magnetic stand and removed with a 10 μ L gun.

10) And opening the cover to dry the magnetic beads for about 5-10min until the ethanol is completely volatilized.

11) Add 12. mu. LddH₂Eluting with O, mixing, standing at room temperature for 3min, placing on magnetic frame for 3min, and recovering 13 μ L product.

The labchip assay was performed on 1. mu.L of the recovered product (ligation product), and the assay results are shown in FIG. 1, in which FIG. 1 shows that the major band of 4 samples after the linker addition was 320 bp. From the results of 4 samples, it can be seen that the End Prep Mix can reduce the stable disruption of genome to a specific size fragment.

4. Library amplification

And carrying out PCR amplification on the recovered product. The PCR amplification reaction system was prepared as shown in table 4:

TABLE 4

Components	Volume of
		Amplification module	12.5ul
Ligation product	11.5ul
		Forward primer (10uM)	0.5ul
Reverse primer (10uM)	0.5ul
		Total of	25ul

A forward primer: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO:6)

Reverse primer: 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO:7)

The PCR amplification reaction conditions are shown in Table 5:

TABLE 5

5. Purification of

The reaction product was purified using VAHTS DNA Clean Beads:

(1) after the Beads were equilibrated to room temperature, VAHTS DNA Clean Beads were vortexed and mixed well.

(2) The ligated PCR system (25ul) was transferred to a 1.5mL EP tube, and 1.8X (45ul) magnetic beads were added thereto, and the mixture was pipetted and mixed 10 times, and allowed to stand at room temperature for 3 min.

(3) Place 1.5mL of EP tube on magnetic stand, let stand for 2-3min, and remove the supernatant.

(4) The beads were washed on the shelf with freshly prepared 200ul 80% ethanol.

(5) Repeat step 4

(6) After removing the ethanol, the mixture was centrifuged instantaneously, placed on a magnetic stand again, and then removed with a 10ul gun.

(7) And opening the cover to dry the magnetic beads for about 5-10min until the ethanol is completely volatilized.

(8) 1.5ml was removed from the magnetic rack and eluted: adding 22ul ddH2O, vortex vibrating or gently blowing and mixing with a pipette, standing at room temperature for 3min, centrifuging the EP tube for a short time, standing in a magnetic frame, clarifying the solution,

20ul of the supernatant was removed to a new 1.5ml EP tube.

1ul of purified and purified product after PCR amplification is taken for labchip detection, the detection result is shown in figure 2, and figure 2 shows that the main band of the product after 4 samples are added with joints for PCR amplification is 320 bp. Because the primer used is matched with the sequence in the joint in a complementary way, the segment can be effectively amplified according to the amplification result, so the joint can be effectively added by using the Ligation Mix, and the segment size is consistent after the joint is interrupted by 4 samples.

6. Quantitative on-machine sequencing

Quantitatively qualified library was used for sequencing by Illumia sequencer.

In the above related steps, preferably, the amount of template DNA is in the range of 15ng-50ng, and OD260/OD280 is 1.8-2.0. The interruption range is 150-200bp

Recommended joint Adapter: the molar ratio of Input DNA is between 100:1 and 600: 1. Excessive Adapter input may result in Adapter or Adapter Dimer residue; insufficient input will affect ligation efficiency and result in reduced library yield. Adapter working concentration 25uM, template molar number (pmol) ≈ Input DNA mass (ng)/[ 0.66. Input DNA average length (bp) ].

The amount of beads directly affects the lower limit of the length of DNA that can be purified. The higher the multiplier, the shorter the lower limit of the length of DNA that can be purified; conversely, the longer. For example: the 1x magnetic beads can only purify DNA longer than 250bp efficiently, and shorter DNA can be lost in a large amount in the purification process; after increasing to 1.8x, 150bp DNA can be purified efficiently.

The eluted product can be stably stored for one week at 4 ℃; the temperature should be kept at-20 deg.C for long term storage to avoid unnecessary repeated freezing and thawing.

Example 3

This example provides the use of the kit of example 1 in library construction 2. In this example, 12 parallel experiments were performed, the used samples were N5, N6, N13, N14, N21, N22, N23, N24, O5, O6, O11, and O12 genomes, and the genome DNA needs to be preliminarily evaluated for genome quality before fragmentation, so as to ensure that the genome exists in a solvent free of metal ion chelating agents or other salts, and the genome integrity is detected by gel electrophoresis. The specific steps of library construction are as follows:

DNA fragmentation, end repair and dA tailing

This step fragments the DNA while filling in the ends of the fragmented DNA, phosphorylates it at the 5 'end and adds a dA tail at the 3' end.

The reaction systems of different samples are the same, and as shown in Table 6, the reaction time at 32 ℃ is different, and is respectively 20min, 22min, 25min, 30min, 35min and 40 min.

The sample types and corresponding reaction times were as follows:

N5:50ng，32℃20min；N6：50ng，32℃20min；

N13:50ng，32℃22min；N14:50ng，32℃22min；

N21:50ng，32℃25min；N22:50ng，32℃25min；

N23:50ng，32℃35min；N24:50ng，32℃35min；

O5:50ng，32℃30min；O6:50ng，32℃30min；

O11:50ng，32℃40min；O12:50ng，32℃40min。

TABLE 6

H2O	14ul
		End Prep Mix	4ul
DNA(25ng/ul)	2ul
		Total	20ul

The PCR tube was placed in a PCR apparatus, and the reaction conditions were set as shown in Table 7: :

TABLE 7

Temp (temperature)	Time (Time)
		105 deg.C thermal cover	On
32℃	(20，22，25，30，35，40)min
		65℃	10min
4℃	Hold

And immediately entering the next operation after the reaction is finished.

Products after the reaction are detected by using Labchip, the detection result is shown in figure 3, and samples corresponding to the highest-lowest main peaks in a detection result graph are N5, N6, N13, N14, N21, N22, N23, N24, O5, O6, O11 and O12. As can be seen from FIG. 3, the breaking time is 20min-40min, the breaking temperature is 32 ℃, the fragmenting enzyme effectively breaks the genome DNA, the broken fragment main band is about 280bp, the different breaking time has little influence on the breaking size of the genome breaking fragment, and the size fluctuation is about 20 bp.

2. Joint connection

After the reaction of step 1, a linker ligation system was prepared in a PCR tube as shown in Table 8:

TABLE 8

Reaction conditions are as follows: standing at room temperature for 15 min.

3. Purification of

The reaction product was purified using VAHTS DNA Clean Beads, the purification procedure is as follows:

12) the adaptor-ligated system was transferred to a 1.5mL EP tube, 0.8X (40. mu.L) magnetic beads were added, and the mixture was pipetted and mixed 10 times, and allowed to stand at room temperature for 3 min.

13) Place 1.5mL of EP tube on magnetic stand, let stand for 2-3min, and remove the supernatant.

14) Freshly prepared 200. mu.L of 80% ethanol was used for bead wash on the shelf.

15) After removal of the ethanol, it was centrifuged instantaneously, placed on a magnetic stand and removed with a 10 μ L gun.

16) And opening the cover to dry the magnetic beads for about 5-10min until the ethanol is completely volatilized.

17) Add 22. mu.L ddH₂Eluting with O, mixing, standing at room temperature for 3min, placing on magnetic frame for 3min, recovering 20 μ L product, adding 1.2x (24ul) magnetic bead, blowing and mixing for 10 times, and standing at room temperature for 3 min.

18) Place 1.5mL of EP tube on magnetic stand, let stand for 2-3min, and remove the supernatant.

19) Freshly prepared 200. mu.L of 80% ethanol was used for bead wash on the shelf.

20) After removal of the ethanol, it was centrifuged instantaneously, placed on a magnetic stand and removed with a 10 μ L gun.

21) And opening the cover to dry the magnetic beads for about 5-10min until the ethanol is completely volatilized.

22) Add 12. mu. LddH₂Eluting with O, mixing, standing at room temperature for 3min, placing on magnetic frame for 3min, and recovering 10 μ L product.

The recovered product can be stably stored at 4 ℃ for one week. The long-term preservation is carried out at the temperature of-20 ℃, and unnecessary repeated freezing and thawing is avoided.

4. Sequence Capture, sequencing on machine

The qualified library was quantitated for sequence capture and sequenced using a single molecule sequencer.

Probes (SEQ ID NO:8) were immobilized on the chip using the method disclosed in the specification of published patent application CN201510501968.7, and the prepared library was diluted with 3X SSC hybridization solution and then hybridized with the probes immobilized on the chip. The number of hybridizations of the linker sequence to the probe was then determined from the signal of Cy 3.

SEQ ID NO:8：5’-TTTTTTTTTTTCCTTGATACCTGCGACCATCCAGTTCCACTCAGATGTGTATAAGAGACAG-3’。

The procedure for library chip hybridization was as follows:

(1) chip selection: the base glass of the chip used was an epoxy-modified glass chip of SCHOTT company, and the probe shown in Seq ID No.4 was immobilized by a method of reacting an amino group on the probe with an epoxy group on the surface of the chip, for example, as disclosed in published patent application No. CN201811191589.2, and the probe density was about 18000Dot/FOV in a 110X 110. mu.M area, that is, 18000 bright spots in a 110X 110. mu.M field of view.

(2) Preparing a hybridization solution: as shown in Table 3, the hybridization solution was prepared using 20 XSSC buffer (Sigma, # S6639-1L) at a final concentration of 3 XSSC, and the library at a final concentration of 1nM in a total volume of 40. mu.L. The prepared hybridization solution is denatured at 95 ℃ for 2min and rapidly cooled on ice.

TABLE 3

20 XSSC buffer	6μL
		Libraries	Final concentration of 1nM
Enucleated acid water	Make up to 40. mu.L

(3) And (3) quickly loading the denatured hybridization solution onto a chip, and then placing the chip at 55 ℃ for 30min to hybridize the library and the probe on the surface of the chip.

(4) The chip was washed sequentially with 3 XSSC, 1 XSSC, and 0.1 XSSC.

Sequencing the library captured by hybridization using the GenoCare third generation sequencing platform.

The above-described preferred embodiments are not intended to limit the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention.

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Claims (10)

1. A method of processing a DNA sample comprising fragmenting, end-repairing and tail-adding said DNA in a first pre-mix system to obtain a first product; the first pre-mix system includes a pre-mixed enzyme that includes a DNA-fragmenting enzyme, a polynucleotide kinase, and a DNA polymerase.

2. The method of claim 1, wherein the DNA is not less than 5 Kbp;

optionally, the first product is 100-400 bp; optionally, the size of the first product is 100-300 bp;

optionally, in the first pre-mixing system, the content of the DNA is 10 ng-60 ng;

optionally, in the first pre-mixing system, the content of the DNA is 15ng-50 ng.

3. The method of any of claims 1-2, characterized in that said first pre-mix system further comprises Mg²⁺Said Mg²⁺The concentration of (A) is 60 mM-100 mM;

optionally, said Mg²⁺The concentration of (A) is 60 mM-75 mM;

optionally, the DNA polymerase is selected from at least one of DNA polymerase I and Taq DNA polymerase;

optionally, the DNA polymerase consists of Klenow fragment of DNA polymerase I and Taq DNA polymerase;

optionally, the polynucleotide kinase is a T4 polynucleotide kinase.

4. The method of claim 3, characterized in that in said first pre-mix system, said DNA fragmenting enzyme is present in an amount of at least 2 times that of said polynucleotide kinase;

optionally, in the first pre-mix system, the DNA fragmenting enzyme, the polynucleotide kinase, the Klenow fragment of DNA polymerase I, and the Taq DNA polymerase are present in a ratio of [25,35]: 6,10]: 1, 3: 3,5 ].

5. The method of any of claims 1-4, further comprising providing at least one terminus of the first product with a predetermined sequence in a second pre-mix system to obtain a second product; the second pre-mix system comprises the first pre-mix system and the preset sequence;

optionally, the second pre-mix system further comprises a ligase;

optionally, the ligase is T4 DNA ligase.

6. The method of claim 5, wherein the predetermined sequence is a double-stranded DNA consisting of a first strand and a second strand that are complementary, the predetermined sequence having at least one single-stranded end;

optionally, the 5' end of the first strand has a phosphate group;

optionally, the 3' end of the second strand does not have a hydroxyl group;

optionally, 80 ℃ or more (Tm1-Tm2) or more is 10 ℃ or more and 90 ℃ or more Tm1 or more is 50 ℃ or more, Tm1 is the dissolution temperature of the first chain, and Tm2 is the dissolution temperature of the second chain;

optionally, the Tm1 is 71 ℃, the Tm2 is 45.6 ℃;

optionally, the predetermined sequence is selected from SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO:1 and SEQ ID NO:3 and/or SEQ ID NO:4 and SEQ ID NO: 5.

7. a method for determining a DNA sequence, comprising:

treating said DNA with the method of any one of claims 1-4 to obtain said first product;

treating said first product by a method according to any one of claims 5 to 6 to obtain said second product;

attaching the second product to a surface of a solid substrate and sequencing the second product to determine at least a portion of the sequence of the DNA.

8. The method of claim 7, characterized in that the second product is provided with an optically detectable label;

optionally, the second product carries a fluorescent molecule;

optionally, the solid phase substrate has a probe on its surface, the second product is attached to the solid phase substrate surface through the probe, and the probe has a length of 20-80 nt;

optionally, the predetermined sequence is double-stranded DNA consisting of a first strand and a second strand that are complementary, the predetermined sequence having a single-stranded end, the single-stranded end being located on the first strand;

optionally, the first strand is fully complementary to the probe, the first strand being no longer than the probe;

optionally, Tm2-5 ℃ T. ltoreq. Tm1-5 ℃ T is the reaction temperature for attaching the second product to the surface of the solid substrate, Tm1 is the dissolution temperature of the first strand, Tm2 is the dissolution temperature of the second strand;

optionally, the method further comprises denaturing the second product prior to attaching the second product to the surface of the solid phase matrix.

9. A kit for carrying out the method of any one of claims 1 to 6 or for carrying out the method of any one of claims 7 to 8, comprising said first pre-mix system.

10. The kit of claim 9, characterized by further comprising said second pre-mix system;

optionally, for performing the method of any one of claims 7-8, further comprising a probe by which the second product is attached to the surface of the solid substrate, the probe having a length of 20-80 nt;

optionally, the probe is complementary to the first strand, the probe is not shorter than the first strand, the predetermined sequence is double-stranded DNA, and consists of the first strand and the second strand that are complementary, the predetermined sequence has a single-stranded end, and the single-stranded end is located on the first strand.

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