CN111005074A - DNA library construction kit based on illumina sequencing platform, library construction method and application - Google Patents
DNA library construction kit based on illumina sequencing platform, library construction method and application Download PDFInfo
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Abstract
The invention provides a DNA library construction kit based on an illumina sequencing platform, a library construction method and application, wherein the kit comprises a P7 joint, a P5 joint, an enzyme composition or a buffer solution, and the enzyme composition comprises any one or a combination of at least two of a terminal repair enzyme, a PCR amplification enzyme or a T4DNA ligase; the buffer comprises a terminal repair buffer and/or a ligation buffer. The kit disclosed by the invention adopts a commercially available reagent to prepare a reaction solution, so that the library construction cost is reduced, the reaction procedure is simplified, the terminal repair reaction system and the A adding reaction system are combined into one system, the reaction time is shortened, and the enzyme ligation efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of sequencing, and relates to a DNA library construction kit based on an illumina sequencing platform, a library construction method and application.
Background
At present, the library building Kit suitable for the second generation of the illumina sequencing platform on the market mainly comprises an illumina original Kit and a KAPA Hyper Prep Kit
platformis, IDT NanoPrep DNA library Kit and NEBNext Ultra II DNA Kit. The main process for constructing the library by adopting the kit is as follows: carrying out fragmentation treatment on a genome; filling ends of the fragmented DNA; adding A at two ends of the filled fragment for reaction; adding adapters at two ends of the fragment by adopting a TA connection mode (the NEBNext Ultra II DNA Kit is that U-shaped adapters are added at two ends of the fragment, and the U-shaped adapters are opened by using USER enzyme); capturing fragments by using AMPure XP magnetic beads to obtain target fragments added with linkers; after PCR enrichment, the PCR product is purified by AMPure XP magnetic beads and then is discharged from the library.
However, the library construction kit has the following defects in constructing the library: (1) the cost of constructing the warehouse is high: the finished product kit is high in price, so that the sequencing cost is greatly increased; (2) the experimental procedure is relatively time consuming: the fragmented DNA is filled in at the ends and the A addition reaction is carried out on both ends of the filled-in fragments, which is carried out separately and takes a long time, for example, KAPAhyper Prep Kit
platformis takes about 1 hour to perform this step; (3) the flow is tedious: the joints added in the NEB kit are U-shaped joints, the U-shaped joints are changed into Y-shaped joints by using USER enzyme after the joints are added, and the experimental process is relatively complicated; (4) the amplification efficiency is low: and for the samples with lower sample size, the database building power is low.
Therefore, the existing kit still has difficulty in meeting the demand of diversification of DNA library construction in the market, and a new library construction kit with low cost, short time consumption, concise flow and high success rate is needed.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a DNA library construction kit based on an illumina sequencing platform, a library construction method and application.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a DNA library construction kit comprising a P7 linker and a P5 linker;
the nucleic acid sequence of the P7 joint is shown as SEQ ID NO 1-5;
the nucleotide sequence of the P5 joint is shown as SEQ ID NO 6-10;
SEQ ID NO:1：
5’-/Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTGATCGTATCTCGTATGCCGTCTTCTGCTTG-3’；
SEQ ID NO:2：
5’-/Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTCTCGAATCTCGTATGCCGTCTTCTGCTTG-3’；
SEQ ID NO:3：
5’-/Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGAGCTAGATCTCGTATGCCGTCTTCTGCTTG-3’；
SEQ ID NO:4：
5’-/Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGACGATATCTCGTATGCCGTCTTCTGCTTG-3’；
SEQ ID NO:5：
5’-/Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTCGAATCTCGTATGCCGTCTTCTGCTTG-3’；
SEQ ID NO:6：
5'-AATGATACGGCGACCACCGAGATCTACACATATGCGCACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3’；
SEQ ID NO:7：
5'-AATGATACGGCGACCACCGAGATCTACACTGGTACAGACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3’；
SEQ ID NO:8：
5'-AATGATACGGCGACCACCGAGATCTACACAACCGTTCACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3’；
SEQ ID NO:9：
5'-AATGATACGGCGACCACCGAGATCTACACTAACCGGTACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3’；
SEQ ID NO:10：
5'-AATGATACGGCGACCACCGAGATCTACACGAACATCGACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3’。
in the invention, s in the nucleic acid sequence shown as SEQ ID NO. 6-10 represents a base which is subjected to thio modification.
Preferably, the kit further comprises an enzyme composition and/or a buffer.
Preferably, the enzyme composition comprises any one of or a combination of at least two of a terminal repair enzyme, a PCR amplification enzyme, or a T4DNA ligase.
Preferably, the buffer comprises a terminal repair buffer and/or a ligation buffer.
Preferably, the end-repair enzyme comprises any one of or a combination of at least two of T4 polynucleotide kinase, T4DNA polymerase, Taq DNA polymerase or Klenow fragment.
Preferably, the working concentration of the T4 polynucleotide kinase is 0.1-0.4U/. mu.L, such as 0.1U/. mu.L, 0.2U/. mu.L, 0.3U/. mu.L or 0.4U/. mu.L.
Preferably, the working concentration of the T4DNA polymerase is 0.05-0.09U/. mu.L, for example, 0.05U/. mu.L, 0.06U/. mu.L, 0.07U/. mu.L, 0.08U/. mu.L or 0.09U/. mu.L.
Preferably, the working concentration of the Taq DNA polymerase is 0.03-0.07U/. mu.L, for example, 0.03U/. mu.L, 0.04U/. mu.L, 0.05U/. mu.L, 0.06U/. mu.L or 0.07U/. mu.L.
Preferably, the Klenow fragment working concentration is 0.01-0.03U/. mu.L, for example, 0.01U/. mu.L, 0.02U/. mu.L or 0.03U/. mu.L.
Preferably, the PCR amplification enzyme comprises Q5 hot start DNA polymerase.
Preferably, the working concentration of the Q5 hot start DNA polymerase is 1X.
Preferably, the working concentration of the T4DNA ligase is 10-30U/. mu.L, for example, 10U/. mu.L, 15U/. mu.L, 20U/. mu.L, 25U/. mu.L or 30U/. mu.L.
Preferably, the end repairing buffer comprises 10 XT 4DNA ligase buffer, dNTP mixed solution, PEG8000 and ultrapure water.
Preferably, the 10 XT 4DNA ligase buffer comprises ATP,Tris-HCl、MgCl2DTT and ultrapure water.
Preferably, the working concentration of the 10 XT 4DNA ligase buffer is 0.8-2X, and may be, for example, 0.8X, 1X, 1.2X, 1.5X, 1.8X or 2X.
Preferably, the dNTP mixture has a working concentration of 0.3 to 0.6mM, for example, 0.3mM, 0.4mM, 0.5mM, or 0.6 mM.
Preferably, the working concentration of the PEG8000 is 5 to 10 wt%, for example, may be 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt% or 10 wt%.
Preferably, the ligation buffer comprises ATP, Tris-HCl, MgCl2DTT, PEG8000, DMSO and ultrapure water.
Preferably, the working concentration of ATP is 0.1-0.5 mM, and may be, for example, 0.1mM, 0.2mM, 0.3mM, 0.4mM, or 0.5 mM.
Preferably, the working concentration of Tris-HCl is 0.01-0.05M, such as 0.01M, 0.02M, 0.03M, 0.04M or 0.05M.
Preferably, the MgCl2The working concentration of (C) is 0.001-0.003M, and may be, for example, 0.001M, 0.002M or 0.003M.
Preferably, the working concentration of DTT is 0.001-0.003M, such as 0.001M, 0.002M or 0.003M.
Preferably, the working concentration of the PEG8000 is 5 to 10 wt%, for example, may be 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt% or 10 wt%.
Preferably, the working concentration of DMSO is 0.8% to 2%, and may be, for example, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, or 2%.
Preferably, the kit further comprises PCR primers.
Preferably, the nucleic acid sequence of the PCR primer is shown as SEQ ID NO. 11-12;
SEQ ID NO:11：AATGATACGGCGACCACCGA；
SEQ ID NO:12：CAAGCAGAAGACATACGA。
in a second aspect, the present invention provides a method of library construction using a kit according to the first aspect.
Preferably, the method comprises the steps of:
(1) carrying out ultrasonic treatment on the genome DNA to obtain fragmented genome DNA;
(2) performing end repair and A addition reaction on the fragmented genome DNA by using end repair enzyme and end repair buffer solution;
(3) performing P7 and P5 joint connection reaction on the end-repair and A product by adopting T4DNA ligase and a connection buffer solution;
(4) purifying the connecting product by using magnetic beads;
(5) and performing PCR amplification on the purified product by adopting Q5 hot start DNA polymerase and PCR primers to construct the DNA library.
Preferably, the power of the ultrasonic treatment in the step (1) is 50-55W, for example, 50W, 51W, 52W, 53W, 54W or 55W.
Preferably, the temperature of the ultrasonic treatment in the step (1) is 15 to 25 ℃, for example, 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃ or 25 ℃.
Preferably, the time of the ultrasonic treatment in the step (1) is 6-12 min, for example, 6min, 7min, 8min, 9min, 10min, 11min or 12 min.
Preferably, the conditions of the end repairing and A adding reaction in the step (2) are that the temperature is kept at 20-25 ℃ for 5-15 min, and the temperature is kept at 70-75 ℃ for 5-15 min.
Preferably, the condition of the joint connection reaction in the step (3) is that the temperature is kept at 20-25 ℃ for 10-20 min.
Preferably, the ratio of P7 to P5 in step (3) is 1: 1.
Preferably, the PCR amplification condition in the step (5) is that the temperature is kept at 95-98 ℃ for 40-50 s; keeping the temperature at 95-98 ℃ for 10-20 s, keeping the temperature at 55-60 ℃ for 30-40 s, keeping the temperature at 70-75 ℃ for 30-40 s, and circulating for 5-10 times; keeping the temperature of 70-75 ℃ for 5-10 min; storing at 4 ℃.
In a third aspect, the invention provides a use of the kit according to the first aspect for constructing an Illumina sequencing library.
Compared with the prior art, the invention has the following beneficial effects:
the kit disclosed by the invention adopts a commercially available reagent to prepare a reaction solution, so that the library construction cost is reduced, the reaction procedure is simplified, the terminal repair reaction system and the A adding reaction system are combined into one system, the reaction time is shortened, and the enzyme ligation efficiency is improved.
Detailed Description
To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 fragmentation of genomic DNA
In this embodiment, after quantifying genomic DNA by using Qubit3.0, 300ng of a sample is added into a 0.2mL PCR tube, 0.1 XTE buffer is added to complement to 55 μ L, a tube cover is marked with a sample number, and the sample number is fully mixed and centrifuged; opening the ultrasonic cleaning instrument, setting the ultrasonic time of the ultrasonic cleaning instrument to be 5min, sequentially placing 0.2mL PCR tubes filled with samples around the ultrasonic sounding position, and breaking 9 samples at a time; after the PCR tube is ensured to be clamped, pure water is injected to the 2/3 scale of the ultrasonic cleaning instrument, a start button is clicked to perform ultrasonic interruption, and the parameter setting is shown in table 1; after the sonication was completed, the fragmented DNA was purified with AMpure XP magnetic beads and dissolved in 39.5. mu.L NF water.
TABLE 1 sonication parameters
| Expected fragment length (bp) | Power (W) | Temperature (. degree.C.) | Ultrasonic time(s) |
| 350 | 55 | 20 | 360 |
| 250 | 55 | 20 | 720 |
Example 2 end repair and A addition reactions
Preparing a tail end repairing reaction solution according to the table 2, and finishing tail end repairing of fragmented DNA and A adding reaction; and (3) blowing and beating the mixture uniformly by using a liquid transfer device, centrifuging the mixture for a short time, collecting the reaction liquid to the bottom of the tube, and performing terminal repair and A addition reaction according to the conditions in the table 3.
TABLE 2 end repair and A addition reaction System
| Reagent | Volume (μ L) |
| Fragmenting DNA | 37 |
| End repair buffer | 10 |
| End repair enzyme | 3 |
TABLE 3 end repair and A addition reaction conditions
| Temperature of | Reaction time |
| 22℃ | 10min |
| 72℃ | 10min |
| 4℃ | ∞ |
EXAMPLE 3 linker ligation reaction
Preparing a linker ligation reaction solution according to table 4 to complete linker ligation of fragmented DNA, wherein a linker group working solution is a self-synthesized mixed sequence of P5(SEQ ID NO: 6-10), P7 linker sequence (SEQ ID NO: 1-5) with a molar ratio of 1:1 and diluted to 10 mu M; and (4) blowing and beating the mixture uniformly by using a liquid transfer device, centrifuging the mixture for a short time, collecting the reaction liquid to the bottom of the tube, and performing joint connection reaction according to the table 5.
TABLE 4 linker ligation reaction System
| Reagent | Volume (μ L) |
| End-repair plus A product | 50 |
| Ligation buffer | 10 |
| T4DNA ligase | 2 |
| Working fluid of joint set (10 mu M) | 3 |
TABLE 5 linker ligation reaction conditions
| Temperature of | Reaction time |
| 22℃ | 15min |
| 4℃ | ∞ |
EXAMPLE 4 ligation product purification
Adding 0.8 times of magnetic beads into the joint connection product, fully and uniformly mixing, standing at room temperature for 5min, placing the PCR tube in a magnetic frame for about 3-5 min to enable the magnetic beads to be completely adsorbed and the solution to be clarified, and carefully removing the supernatant; add 200 μ L of freshly prepared 80% ethanol to rinse the beads, incubate at room temperature for 30s, carefully remove the supernatant, and repeat once again; after the magnetic beads are dried, adding22.5. mu. LddH2And O elution, gently and uniformly mixing by using a pipettor, standing at room temperature for 5min, standing in a magnetic frame, standing for 3-5 min after the solution is clarified, and transferring 20 mu L of supernatant (without touching magnetic beads) to a new PCR tube.
Example 5 PCR enrichment
Preparing PCR amplification reaction solution according to the sequence shown in the SEQ ID NO. 11-12; and (3) blowing and beating the mixture by using a pipette, centrifuging the mixture for a short time to collect reaction liquid to the bottom of the tube, placing the mixed PCR tube on a PCR instrument, setting the temperature of a hot cover to be 105 ℃, and carrying out reaction according to the following table 7.
TABLE 6 PCR amplification System
| Reagent | Volume (μ L) |
| Purified linker ligation product | 20 |
| Primer pair | 5 |
| 2 XQ 5 Hot Start DNA polymerase | 25 |
TABLE 7 PCR amplification conditions
Example 6 PCR product purification
Adding 0.8 times of magnetic beads into the PCR amplification product, fully mixing uniformly, standing at room temperature for 5min, placing the PCR tube in a magnetic frame for about 3-5 min to enable the magnetic beads to be completely adsorbed and obtain a solutionClear and carefully remove the supernatant; add 200 μ L of freshly prepared 80% ethanol to rinse the beads, incubate at room temperature for 30s, carefully remove the supernatant, and repeat once again; after the beads were dried, 20. mu. LddH was added2And O elution, gently and uniformly mixing by using a pipette, standing at room temperature for 5min, standing in a magnetic frame, standing for 3-5 min after the solution is clarified, and transferring 18 mu L of supernatant (without touching magnetic beads) to a new PCR tube.
Example 7 library quality testing machine
Taking 1 microliter of purified library, quantifying by using the Qubit3.0, detecting the fragment size of the library by agarose gel electrophoresis, carrying out qPCR quantitative quality inspection on the qualified library in the primary quality inspection, and operating the qualified library on a computer.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Jiangxi sea Prolos medical testing laboratory Co., Ltd
<120> DNA library construction kit based on illumina sequencing platform, library construction method and application
<130>20191212
<160>12
<170>PatentIn version 3.3
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Claims (10)
1. A DNA library construction kit comprising a P7 linker and a P5 linker;
the nucleic acid sequence of the P7 joint is shown as SEQ ID NO 1-5;
the nucleic acid sequence of the P5 joint is shown as SEQ ID NO 6-10.
2. The kit of claim 1, wherein the kit further comprises an enzyme composition and/or a buffer;
preferably, the enzyme composition comprises any one of or a combination of at least two of a terminal repair enzyme, a PCR amplification enzyme, or a T4DNA ligase;
preferably, the buffer comprises a terminal repair buffer and/or a ligation buffer.
3. The kit of claim 2, wherein the end-repair enzyme comprises any one of or a combination of at least two of T4 polynucleotide kinase, T4DNA polymerase, Taq DNA polymerase, or Klenow fragment;
preferably, the working concentration of the T4 polynucleotide kinase is 0.1-0.4U/. mu.L;
preferably, the working concentration of the T4DNA polymerase is 0.05-0.09U/muL;
preferably, the working concentration of the Taq DNA polymerase is 0.03-0.07U/mu L;
preferably, the working concentration of the Klenow fragment is 0.01-0.03U/. mu.L.
4. The kit of claim 2, wherein the PCR amplification enzymes comprise Q5 hot start DNA polymerase;
preferably, the working concentration of the Q5 hot start DNA polymerase is 1 ×;
preferably, the working concentration of the T4DNA ligase is 10-30U/. mu.L.
5. The kit of claim 2, wherein the end-repair buffer comprises 10 xt 4DNA ligase buffer, dNTP mix, PEG8000 and ultrapure water;
preferably, the 10 XT 4DNA ligase buffer comprises ATP, Tris-HCl, MgCl2DTT and ultrapure water;
preferably, the working concentration of the 10 XT 4DNA ligase buffer solution is 0.8-2 ×;
preferably, the working concentration of the dNTP mixed solution is 0.3-0.6 mM;
preferably, the working concentration of the PEG8000 is 5-10 wt%.
6. The kit of claim 2, wherein the ligation buffer comprises ATP, Tris-HCl, MgCl2DTT, PEG8000, DMSO and ultrapure water;
preferably, the working concentration of ATP is 0.1-0.5 mM;
preferably, the working concentration of the Tris-HCl is 0.01-0.05M;
preferably, the MgCl2The working concentration of (A) is 0.001-0.003M;
preferably, the working concentration of the DTT is 0.001-0.003M;
preferably, the working concentration of the PEG8000 is 5-10 wt%;
preferably, the working concentration of DMSO is 0.8% to 2%.
7. The kit of any one of claims 1 to 6, wherein the kit further comprises PCR primers;
preferably, the nucleic acid sequence of the PCR primer is shown as SEQ ID NO. 11-12.
8. A method for library construction using a kit according to any one of claims 1 to 7;
preferably, the method comprises the steps of:
(1) carrying out ultrasonic treatment on the genome DNA to obtain fragmented genome DNA;
(2) performing end repair and A addition reaction on the fragmented genome DNA by using end repair enzyme and end repair buffer solution;
(3) performing P7 and P5 joint connection reaction on the end-repair and A product by adopting T4DNA ligase and a connection buffer solution;
(4) purifying the connecting product by using magnetic beads;
(5) and performing PCR amplification on the purified product by adopting Q5 hot start DNA polymerase and PCR primers to construct the DNA library.
9. The method according to claim 8, wherein the power of the ultrasonic treatment in the step (1) is 50-55W;
preferably, the temperature of the ultrasonic treatment in the step (1) is 15-25 ℃;
preferably, the time of the ultrasonic treatment in the step (1) is 6-12 min;
preferably, the conditions of the end repairing and A adding reaction in the step (2) are that the temperature is kept at 20-25 ℃ for 5-15 min, and the temperature is kept at 70-75 ℃ for 5-15 min;
preferably, the condition of the joint connection reaction in the step (3) is that the temperature is kept at 20-25 ℃ for 10-20 min;
preferably, the ratio of P7 and P5 in step (3) is 1: 1;
preferably, the PCR amplification condition in the step (5) is that the temperature is kept at 95-98 ℃ for 40-50 s; keeping the temperature at 95-98 ℃ for 10-20 s, keeping the temperature at 55-60 ℃ for 30-40 s, keeping the temperature at 70-75 ℃ for 30-40 s, and circulating for 5-10 times; keeping the temperature of 70-75 ℃ for 5-10 min; storing at 4 ℃.
10. Use of the kit of any one of claims 1-7 for constructing an Illumina sequencing library.
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