CN106567132B - Library building method suitable for simplified genome sequencing - Google Patents
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Abstract
The invention provides a library construction method for simplifying genome sequencing, which comprises the steps of respectively connecting a universal joint and a bar code joint at two ends of an enzyme digestion product to obtain a connection product and performing pool mixing; adding 1.2-1.4 times of magnetic beads into the connection product after the mixing tank for first purification to obtain a first purified product; adding 0.8-0.9 volume times of magnetic beads into the first purified product to carry out second purification to obtain a second purified product; performing PCR amplification on the second purified product to obtain a PCR product; adding 1.2-1.4 times of magnetic beads into the PCR product for third purification to obtain a third purified product; and adding 0.8-0.9 times of magnetic beads into the third purified product to carry out fourth purification to obtain a simplified genome sequencing library. The method can stably grab the specific length of the genome for high-throughput SNP typing library establishment, reduces the influence caused by short enzyme digestion products in the PCR process, and enables the simplified genome sequencing technology to be better transplanted and popularized in different endonucleases and different species.
Description
Technical Field
The invention relates to the technical field of genome sequencing, in particular to a database construction method suitable for simplifying genome sequencing.
Background
Simplified-genome sequencing (reduced-representation sequencing) is a technique for stably recovering a part of enzyme digestion products for sequencing after the enzyme digestion and breaking of genome DNA by restriction enzymes, and is called as simplified genome sequencing because the recovered products mostly only account for 0.1-5% of the whole genome. Currently, the commonly used simplified genome Sequencing mainly comprises methods such as RAD-Seq (Restriction-site Associated DNA Sequencing), GBS (genotyping By Sequencing), 2b-RAD (IIB Restriction-Associated DNA Sequencing) and the like. Through the selection and combination of different restriction enzymes, the methods are successfully applied to most animals and plants, so that the identification and typing of medium-high flux molecular markers covering the whole genome required by population analysis research becomes possible, and particularly for species with insufficient genome information research or without commercialized high-density SNP chips, the simplified genome sequencing technology is more necessary.
The simple and rapid library construction method is the key for simplifying genome sequencing and can directly influence the reliability and stability of results. The method for constructing the library by the RAD-Seq technology comprises the following steps: firstly, restriction enzyme cuts the DNA enzyme, and then a first joint is connected; then breaking the connected product by using ultrasonic waves, and connecting a second joint; finally, the 300-700bp ligation product is recovered by a gel recovery method. The biggest disadvantage of this method is that the steps of ultrasound disruption and gel recovery cannot guarantee reproducibility between each sample, so that the genomic regions measured by each individual are very different, increasing the difficulty of subsequent data analysis. The 2B-RAD technology is similar to the method for constructing the library by the RAD-Seq technology, and the difference is that the used restriction enzyme is type II type B restriction enzyme (RAD-Seq is type II type A restriction enzyme), the restriction enzyme cuts 8-15bp positions at two sides of the identified sequence, thereby generating a restriction enzyme product with the size of about 30bp, and after connecting a specific joint, the fragment is recovered by a glue recovery method; the disadvantage is that the fragments generated by digestion are too small (about 30bp), and the fragments obtained by digestion at very high density need to be sequenced to detect a proper amount of genetic variation. The GBS technology is developed by Elshire et al of Cannell university, the library construction method is the simplest, DNA is connected with a specific joint after enzyme digestion, and a part of 100-plus 500bp in an enzyme digestion product is selected by controlling the extension time (generally 30 seconds) in the PCR process, so that the aim of simplifying genome sequencing is fulfilled; however, the method has the defects that the amplification efficiency of small fragment enzyme digestion products in the library building process is higher, the small fragment enzyme digestion products can grow faster in the template amplification of a sequencer, and the problems that the usable part of sequencing data is less and the data quality is poor are easily caused.
Because the existing several common simplified genome database building methods have the defects, the existing simplified genome database building technology needs to be improved, and a new method which is simple, convenient and quick, has good repeatability and stably captures the specific length segment of the genome is developed so as to reduce the influence of small fragment enzyme digestion products on the data quality.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a library construction method capable of stably grabbing specific length fragments of a genome for high-throughput SNP typing, and on the premise of not adopting a gel recovery method, the influence caused by short enzyme digestion products in the PCR process is reduced, so that the simplified genome sequencing technology can be better transplanted and popularized in different endonucleases and different species.
To this end, the invention provides a library construction method suitable for simplified genome sequencing, comprising the steps of:
(a) carrying out enzyme digestion on a pig genome by using restriction enzyme;
(b) preparing a universal joint and a bar code joint;
(c) respectively carrying out connection reaction on the universal joint and the bar code joint and the enzyme digestion product to obtain a connection product;
(d) mixing the connecting products in equal proportion in a pool to obtain the connecting products after the pool is mixed;
(e) adding 1.2-1.4 times of magnetic beads into the connection product after the mixing tank for first purification to obtain a first purified product;
(f) adding 0.8-0.9 volume times of magnetic beads into the first purified product to carry out second purification to obtain a second purified product;
(g) performing PCR amplification on the second purified product to obtain a PCR product;
(h) adding 1.2-1.4 times of magnetic beads into the PCR product for third purification to obtain a third purified product;
(i) and adding 0.8-0.9 times of magnetic beads into the third purified product to carry out fourth purification to obtain a simplified genome sequencing library.
The first purification and the third purification have the same steps, and specifically comprise:
adding the magnetic beads, and incubating on a rotator at room temperature for 18-22min to obtain an incubated system; after the incubation is finished, adding 70% ethanol with the volume of 0.9-1.1 times of that of the incubated system, standing for 30-40s, slowly rotating to enable the magnetic beads to move on the wall of the tank, removing supernatant after the solution is clarified, and repeating the step once to obtain a precipitate; wherein, the amount of 70% ethanol added each time is 90-110 μ L relative to 100 μ L of the post-incubation system; adding Low TE into the obtained precipitate, sucking up and down by using a pipettor, oscillating for 10s, centrifuging, standing and clarifying to obtain a supernatant; wherein the addition amount of Low TE is 140-160. mu.L relative to 100. mu.L of the precipitate.
Wherein, the second purification and the fourth purification have the same steps, and specifically comprise:
adding magnetic beads, and incubating at room temperature for 13-16min on a rotary instrument; adding 70% ethanol with the volume of 0.9-1.1 times of the volume of the solution after the incubation is finished, standing for 30-40s, slowly rotating to enable the magnetic beads to move on the wall of the tank, removing supernatant after the solution is clarified, and repeating the step once to obtain a precipitate; adding Low TE into the obtained precipitate, sucking up and down by using a pipettor, oscillating for 10s, centrifuging, standing and clarifying to obtain a supernatant; wherein the amount of Low TE added is 30-50. mu.L per 100. mu.L of the precipitate.
Wherein the magnetic Beads are AMPure XP Beads.
Wherein the universal adaptor and the barcode adaptor both have the same cohesive end sequence as the restriction enzyme.
Wherein, in step (b), the universal linker is a linker consisting of SEQ ID NO:1 and SEQ ID NO:2, wherein the sequence shown in SEQ ID NO:1 is modified by 5' phosphorylation;
the barcode linker is represented by SEQ ID NO:3 and SEQ ID NO:4 to form double-stranded DNA; wherein SEQ ID NO:4, modified by 5' phosphorylation, SEQ ID NO:3 and SEQ ID NO: n and m in 4 represent arbitrary short nucleotide barcode sequences of 5-9bp in length for distinguishing different samples.
Wherein, the general joint annealing system is: 100 μ M of SEQ ID NO 15 μ L; mu.L of 100. mu.M SEQ ID NO 25. mu.L, 5 × Annealing Buffer 10. mu.L, nuclease-free water 30. mu.L; the annealing procedure is as follows: heating to 95 deg.C, cooling to 25 deg.C at a rate of 1 deg.C/min, and maintaining at 25 deg.C for 30 min.
Wherein, the bar code connects annealing system: 35. mu.L of 100. mu.M SEQ ID NO, 45. mu.L of 100. mu.M SEQ ID NO, 10. mu.L of 5 × analizing buffer, and 30. mu.l of nuclease-free water; the annealing procedure is as follows: cooling at 95 deg.C for 3min at 1 deg.C/min until the temperature is reduced to 25 deg.C, and maintaining at 25 deg.C for 30 min.
In the step (c), firstly, mixing a universal joint and a bar code joint according to a ratio of 3:1 to obtain a joint mixture, and then connecting the joint mixture to two ends of an enzyme digestion product; the mixed system is as follows: 3.6 μ L of universal linker, 1.2 μ L of barcode linker, and 195.2 μ L of nuclease-free water.
Wherein, in the step (c), the connection reaction system is: 20 mu L of enzyme digestion product, 8 mu L of 5 XDNA Ligase reaction buffer, 2 mu L of DNA Ligase, 5 mu L of nuclease-free water and 5 mu L of linker mixture; after mixing, placing on PCR, wherein the reaction procedure is as follows: storing at 22 deg.C for 1h and 65 deg.C for 30min at 4 deg.C.
Wherein, in the step (g), the primer pair used for PCR amplification is shown as SEQ ID NO: 5 and SEQ ID NO: and 6.
In the step (g), the PCR reaction system is as follows: 50 μ L of high fidelity PCR premix, 0.5 μ L of second purified product, 10 μ MSEQ ID NO: 51.2 μ L, 10 μ M SEQ ID NO: 61.2. mu.L of nuclease-free water, 7.1. mu.L; the PCR reaction program is: 95 ℃ for 5min, 17 cycles X (95 ℃ for 30s, 62 ℃ for 30s, 68 ℃ for 30s), 72 ℃ for 5min, 4 ℃ storage.
The method provided by the invention adopts 1.2-1.4 times and 0.8-0.9 times of magnetic beads to respectively purify the enzyme digestion product and the PCR product, so that the library building process is simple, convenient and quick, and the library quality is high. On the premise of not adopting a gel recovery method, the influence caused by short enzyme digestion products in the PCR process is reduced, so that the simplified genome sequencing technology can be better transplanted and popularized in different endonucleases and different species.
Drawings
FIG. 1 shows the library effect of optimizing the magnetic bead purification conditions, using 1.0+0.8 times volume purification.
FIG. 2 shows the library effect of optimizing the magnetic bead purification conditions, using 1.1+0.8 times volume purification.
FIG. 3 shows the library effect of optimizing the magnetic bead purification conditions, using 1.2+0.8 times volume purification.
FIG. 4 shows the library effect of optimizing the magnetic bead purification conditions, using 1.3+0.8 times volume purification.
FIG. 5 shows the effect of library purification with 1.4+0.8 times volume optimized magnetic bead purification conditions.
FIG. 6 shows the library effect of optimizing the magnetic bead purification conditions, using 1.3+0.9 times volume purification.
FIG. 7 shows the library effect of optimizing the magnetic bead purification conditions using 1.3+1.0 times volume purification.
FIG. 8 is a simplified genome sequencing data quality profile.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The following examples are provided for illustration only and are not intended to limit the scope of the present invention, and include EcoRI-MspI double-restriction enzyme digestion of pig genomic DNA followed by simplified genomic library construction and sequencing.
Example 1 for illustrating the effect of magnetic bead purification
1. Experimental Material
Genomic DNA of 1 pig ear tissue was extracted using QIAGEN tissue DNA extraction kit, dissolved in TE, and diluted to 50 ng/. mu.L.
2. Design and Synthesis of the following sequences
The universal linker comprises the following two sequences:
oligo1：5’-CGAGATCGGAAGAGCGGGGACTTTAAGC-3’；
oligo2：5’-GATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3’；
the barcode linker comprises the following two sequences:
oligo1：5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGTT-3’；
oligo2：5’-AATTAACAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3’；
the PCR primers comprise the following two sequences:
oligo1：5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
oligo2：
5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAA-3’
3. annealing to prepare universal and bar code connectors
The annealing system is: mu.L of 100. mu.M oligo 15. mu.L, 100. mu.M oligo 25. mu.L, 5 × analizing Buffer 10. mu.L, nuclease-free water 30. mu.L; the annealing procedure is as follows: heating to 95 deg.C, cooling at 1 deg.C/min until the temperature is reduced to 25 deg.C, and maintaining at 25 deg.C for 30 min.
The joint mixture was prepared as follows: 3.6 μ L of universal linker, 1.2 μ L of barcode linker, 195.2 μ L of nuclease-free water, and 200 μ L of total.
4. DNA cleavage
EcoRI enzyme and MspI enzyme are adopted to carry out double enzyme digestion on genome, and the enzyme digestion system is as follows: 200ng of genomic DNA, 0.5. mu.L of EcoRI enzyme, 0.5. mu.L of MspI enzyme, 10 XCutSmart Buffer 2. mu.L, 13. mu.L of nuclease-free water, and 20. mu.L in total. After being uniformly mixed, the enzyme digestion system is placed in a PCR instrument for reaction, and the PCR program is as follows: storing at 37 deg.C for 90min, 65 deg.C for 30min, and 4 deg.C.
5. Connecting joint
The linking system is as follows: 20 mu L of enzyme digestion product, 8 mu L of 5 XDNA Ligase Reaction Buffer, 2 mu L of DNA Ligase, 5 mu L of nuclease-free water and 5 mu L of linker mixture; after mixing, placing on PCR, wherein the reaction procedure is as follows: storing at 22 deg.C for 1 hr, at 65 deg.C for 30min, and at 4 deg.C.
6. PCR amplification of libraries
The PCR reaction system is as follows: 50 muL of high-fidelity PCR premix, 0.5 muL of ligation product, 11.2 muL of 10 muM PCR primer oligo, 21.2 muL of 10 muM PCR primer oligo, and 7.1 muL of nuclease-free water; the PCR reaction program is: 95 ℃ for 5min, 17 cycles X (95 ℃ for 30s, 62 ℃ for 30s, 68 ℃ for 30s), 72 ℃ for 5min, 4 ℃ storage.
7. Purification combination design of magnetic beads
In order to study the optimal magnetic bead addition amount for the first round, the third round, the second round and the fourth round of magnetic bead purification, the following 7 magnetic bead combinations with different proportions are respectively designed and compared:
(1)1.0+0.8，1.1+0.8，1.2+0.8，1.3+0.8，1.4+0.8
(2)1.3+1.0，1.3+0.9
8. library quality inspection
The library was examined for fragment size by 2100, and the optimal purification protocol was judged by the cleanliness of large and small fragment DNA removal.
9. Results
Comparing fig. 1, fig. 2, fig. 3, fig. 4, and fig. 5, it can be seen that the optimal purification effect in the first group is 1.3+0.8, and thus the removal degree and recovery efficiency for large fragments are optimal when the volume of the magnetic beads used in the first and third purification rounds is 1.3 times. In addition, the purification effect of 1.2+0.8 and 1.4+0.8 are better, the difference is that the library recovery efficiency is slightly lower when the ratio of 1.2+0.8 is used, and the removal degree of large fragments is slightly poor when the combination of 1.4+0.8 is used, but the slight difference does not actually affect the overall effect of the library, because the magnetic beads used in the first and third rounds of purification are 1.2-1.4 times of volume.
Comparing the results of fig. 4, 6 and 7, it can be seen that the second round and the fourth round of magnetic beads are purified to 0.8-0.9 times the volume, and the small fragments are removed to the best extent.
It can be seen that the method provided by the present invention uses 1.3 times (or 1.2-1.4 times) volume for the first round and the third round of magnetic bead purification, and uses 0.8-0.9 times volume for the second round and the fourth round of magnetic bead purification, thereby obtaining the best small fragment removal effect.
Example 2 simplified genome sequencing of 24 pigs
1. Experimental Material
Genomic DNA of 24 pig ear tissues was extracted using QIAGEN tissue DNA extraction kit, dissolved in TE, and diluted to 50 ng/. mu.L.
2. The sequences in Table 1 below were designed and synthesized, wherein among the barcode linker sequences, the short sequences with the length of 5-9bp, which are the italicized black parts, were barcode sequences for distinguishing the sequencing data of 24 samples, so that 24 samples could be sequenced by mixing them together.
TABLE 1 sequences required for simplified genomic sequencing of 24 pigs
3. Preparation of Bar code Joint and Universal Joint
Annealing of oligo1 sequence and oligo2 sequence resulted in barcode linker and universal linker. The annealing system is: mu.L of 100. mu.M oligo 15. mu.L, 100. mu.M oligo 25. mu.L, 5 × analizing Buffer 10. mu.L, nuclease-free water 30. mu.L; the annealing procedure is as follows: heating to 95 deg.C, cooling at 1 deg.C/min until the temperature is reduced to 25 deg.C, and maintaining at 25 deg.C for 30 min.
The joint mixture was prepared as follows: 3.6 μ L of universal linker, 1.2 μ L of barcode linker, 195.2 μ L of nuclease-free water, and 200 μ L of total.
4. DNA cleavage
EcoRI enzyme and MspI enzyme are adopted to carry out double enzyme digestion on genome, and the enzyme digestion system is as follows: 200ng of genomic DNA, 0.5. mu.L of EcoRI enzyme, 0.5. mu.L of MspI enzyme, 2. mu.L of 10 XCutSmart Buffer, 13. mu.L of nuclease-free water, and 20. mu.L of the total. After being uniformly mixed, the enzyme digestion system is placed in a PCR instrument for reaction, and the PCR program is as follows: storing at 37 deg.C for 90min, 65 deg.C for 30min, and 4 deg.C.
5. Connecting joint
The linking system is as follows: 20 mu L of enzyme digestion product, 8 mu L of 5 XDNA Ligase Reaction Buffer, 2 mu L of DNA Ligase, 5 mu L of nuclease-free water and 5 mu L of linker mixture; after mixing, placing on PCR, wherein the reaction procedure is as follows: storing at 22 deg.C for 1 hr, at 65 deg.C for 30min, and at 4 deg.C.
6. Isometric mixed ligation product
mu.L of the ligation product was removed from each individual, 120. mu.L in total, and mixed well.
7. Ligation product purification
(1) Adding 1.3 times volume of AMPure XP Beads into the connection product, placing on a rotary instrument, and incubating for 20min at room temperature;
(2) adding 500 μ L70% ethanol (centrifuge tube on magnetic frame), rotating the tube slowly after 30s to make magnetic beads move on the tube wall, and removing supernatant after the solution is clarified;
(3) repeating the step (2) once;
(4) adding 150 mu L of Low TE, sucking and beating for several times by a gun, oscillating for 10s, placing on a magnetic frame after short-time centrifugation, and transferring the supernatant into a new centrifuge tube after clarification;
(5) adding 0.8 times volume of AMPure XP Beads into the connection product, placing on a rotary instrument, and incubating for 15min at room temperature;
(6) adding 500 μ L70% ethanol (centrifuge tube on magnetic frame), rotating the tube slowly after 30s to make magnetic beads move on the tube wall, and removing supernatant after the solution is clarified;
(7) repeating the step (6) once;
(8) adding 50 mu L of Low TE, sucking and beating for several times by a gun, oscillating for 10s, placing on a magnetic frame after short-time centrifugation, and transferring the supernatant into a new centrifuge tube after clarification;
8. PCR amplification
The PCR reaction system is as follows: 50 muL of high-fidelity PCR premix, 0.5 muL of ligation product, 11.2 muL of 10 muM PCR primer oligo, 21.2 muL of 10 muM PCR primer oligo, and 7.1 muL of nuclease-free water; the PCR reaction program is: 95 ℃ for 5min, 17 cycles X (95 ℃ for 30s, 62 ℃ for 30s, 68 ℃ for 30s), 72 ℃ for 5min, 4 ℃ storage.
9. PCR product purification
(1) Adding 1.3 times volume of AMPure XP Beads into the connection product, placing on a rotary instrument, and incubating for 20min at room temperature;
(2) adding 500 μ L70% ethanol (centrifuge tube on magnetic frame), rotating the tube slowly after 30s to make magnetic beads move on the tube wall, and removing supernatant after the solution is clarified;
(3) repeating the step (2) once;
(4) adding 150 mu L of Low TE, sucking and beating for several times by a gun, oscillating for 10s, placing on a magnetic frame after short-time centrifugation, and transferring the supernatant into a new centrifuge tube after clarification;
(5) adding 0.8 times volume of AMPure XP Beads into the connection product, placing on a rotary instrument, and incubating for 15min at room temperature;
(7) adding 500 μ L70% ethanol (centrifuge tube on magnetic frame), rotating the tube slowly after 30s to make magnetic beads move on the tube wall, and removing supernatant after the solution is clarified;
(7) repeating the step (6) once;
(8) adding 50 mu L of Low TE, sucking and beating for several times by a gun, oscillating for 10s, placing on a magnetic frame after short-time centrifugation, and transferring the supernatant into a new centrifuge tube after clarification;
(9) quality control of library
The size of the fragment of the library is detected through 2100, and the library is judged to be qualified through the cleanness of removing large fragment DNA and small fragment DNA, so that the method is suitable for on-machine sequencing.
(10) Illumina Hiseq2500 sequencing
The resulting library was sequenced using the Illumina Hiseq2500 platform, using 100bp paired ends for sequencing. The results show that 82,007,411 reads are obtained in total by sequencing, wherein 73,750,378 reads just fall at the enzyme cutting site, which account for 89.93% of the obtained data, and the quality of the visible library is very good; the base quality of the sequencing data is shown in FIG. 8, and it can be seen that the sequence obtained by sequencing has very high quality, which also indicates that the quality of the library is relatively high.
(11) Stability analysis of database construction
The position of the cleavage fragment detected in each sample on the reference genome is obtained by bioinformatics analysis, and the number of times these cleavage fragments were detected in the samples used is compared. The results showed that on average 427,416 cleavage fragments were detected per sample; by analyzing the number of times of the enzyme fragments reappearing in the samples (table 2), the results show that about 281,509 enzyme fragments can be detected in 20-24 samples, that is, at least 281,509/427,416 × 100% ═ 65.86% of the enzyme fragments detected in each sample are stably captured successfully by the simplified genome library building method, which shows that the library building method of the present invention has very stable results and is suitable for sequencing a large number of samples.
TABLE 2 reproducibility of the determined cleavage sites in 24 samples
The embodiment of the invention only uses EcoRI-MspI to enzyme cut pig genome DNA for library construction and sequencing to show that the method is simple, convenient and quick and has good stability, but the library construction method is also suitable for simplified genome sequencing of other organisms and other endonuclease combinations.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method of pooling for simplified genome sequencing comprising the steps of:
(a) carrying out enzyme digestion on a pig genome by using restriction enzyme;
(b) preparing a universal joint and a bar code joint;
(c) mixing the universal joint and the bar code joint to obtain a joint mixture, and performing a connecting reaction on the joint mixture and a restriction enzyme product to obtain a connecting product;
(d) adding 1.2-1.4 times of magnetic beads into the connection product for first purification to obtain a first purification product;
(e) adding 0.8-0.9 volume times of magnetic beads into the first purified product to carry out second purification to obtain a second purified product;
(f) performing PCR amplification on the second purified product to obtain a PCR product;
(g) adding 1.2-1.4 times of magnetic beads into the PCR product for third purification to obtain a third purified product;
(h) and adding 0.8-0.9 times of magnetic beads into the third purified product to carry out fourth purification to obtain a simplified genome sequencing library.
2. The method according to claim 1, wherein the first and third purification steps are the same, in particular comprising:
adding the magnetic beads, and incubating on a rotator at room temperature for 18-22min to obtain an incubated system; after the incubation is finished, adding 70% ethanol with the volume of 0.9-1.1 times of that of the incubated system, standing for 30-40s, slowly rotating to enable the magnetic beads to move on the wall of the tank, removing supernatant after the solution is clarified, and repeating the step once to obtain a precipitate; wherein, the amount of 70% ethanol added each time is 90-110 μ L relative to 100 μ L of the post-incubation system; adding Low TE into the obtained precipitate, sucking up and down by using a pipettor, oscillating for 10s, centrifuging, standing and clarifying to obtain a supernatant; wherein the addition amount of Low TE is 140-160. mu.L relative to 100. mu.L of the precipitate.
3. The method according to claim 1, wherein the second and fourth purification steps are the same, and in particular comprise:
adding magnetic beads, and incubating at room temperature for 13-16min on a rotary instrument; adding 70% ethanol with the volume of 0.9-1.1 times of the volume of the solution after the incubation is finished, standing for 30-40s, slowly rotating to enable the magnetic beads to move on the wall of the tank, removing supernatant after the solution is clarified, and repeating the step once to obtain a precipitate; adding Low TE into the obtained precipitate, sucking up and down by using a pipettor, oscillating for 10s, centrifuging, standing and clarifying to obtain a supernatant; wherein the amount of Low TE added is 30-50. mu.L per 100. mu.L of the precipitate.
4. A method according to any of claims 1-3, wherein said magnetic beads are AMPure XPBeads.
5. The method of claim 4, wherein in step (b), the universal linker is a linker consisting of SEQ ID NO:1 and SEQ ID NO:2, wherein the sequence shown in SEQ ID NO:1 is modified by 5' phosphorylation;
the barcode linker is represented by SEQ ID NO:3 and SEQ ID NO:4 to form double-stranded DNA; wherein SEQ ID NO:4, modified by 5' phosphorylation, SEQ ID NO:3 and SEQ ID NO: n and m in 4 represent arbitrary short nucleotide barcode sequences of 5-9bp in length.
6. The method of claim 1 or 5, wherein the universal joint annealing system is: 100 μ M SEQ ID NO 15 μ L; mu.L of 100. mu.M SEQ ID NO 25. mu.L, 5 × Annealing Buffer 10. mu.L, nuclease-free water 30. mu.L; the annealing procedure is as follows: heating to 95 deg.C, cooling to 25 deg.C at a rate of 1 deg.C/min, and maintaining at 25 deg.C for 30 min;
the bar code joint annealing system is as follows: 35. mu.L of 100. mu.M SEQ ID NO, 45. mu.L of 100. mu.M SEQ ID NO, 10. mu.L of 5 × analizing buffer, and 30. mu.l of nuclease-free water; the annealing procedure is as follows: cooling at 95 deg.C for 3min at 1 deg.C/min until the temperature is reduced to 25 deg.C, and maintaining at 25 deg.C for 30 min.
7. The method of claim 6, wherein in step (c), the universal linker and the barcode linker are mixed in a ratio of 3:1 to obtain a linker mixture, and the linker mixture is connected to both ends of the enzyme-cleaved product; the mixed system is as follows: 3.6 mu L of universal joint, 1.2 mu L of bar code joint and 195.2 mu L of nuclease-free water, and the mixture is mixed evenly.
8. The method of claim 1 or 7, wherein in step (c), the ligation reaction system is: 20 mu L of enzyme digestion product, 8 mu L of 5 XDNA Ligase Reaction Buffer, 2 mu L of DNA Ligase, 5 mu L of nuclease-free water and 5 mu L of linker mixture; after mixing, placing on PCR, wherein the reaction procedure is as follows: storing at 22 deg.C for 1h and 65 deg.C for 30min at 4 deg.C.
9. The method of claim 8, wherein in step (f), the primer pair used for PCR amplification is as set forth in SEQ ID NO: 5 and SEQ ID NO: and 6.
10. The method of claim 9, wherein in step (f), the PCR reaction system is: 50 μ L of high fidelity PCR premix, 0.5 μ L of second purified product, 10 μ M of SEQ ID NO: 51.2 μ L, 10 μ M SEQ ID NO: 61.2. mu.L of nuclease-free water, 7.1. mu.L; the PCR reaction program is: 5min at 95 ℃; 30s at 95 ℃, 30s at 62 ℃ and 30s at 68 ℃ for 17 cycles; preserving at 72 deg.C for 5min and 4 deg.C.
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| CN111560463B (en) * | 2020-06-15 | 2022-08-26 | 山东丰沃植物研究院有限公司 | Three gala apple specific molecular markers and screening method and application thereof |
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| A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species;Robert J. Elshire等;《PLOS ONE》;20110504;第6卷(第5期);第2页右栏第2段，第3页图1 * |
| Agencourt AMPure XP Information For Use Guide;Agencourt AMPure XP Information For Use Guide;《www.beckmancoulter.com》;20130831;第3-6页 * |
| An Efficient Genotyping Method in Chicken Based on Genome Reducing and Sequencing;Rongrong Liao等;《PLOS ONE》;20150827;第10卷(第8期);第3页第2段至第5段 * |
| Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach;Jesse A. Poland等;《PLOS ONE》;20120228;第7卷(第2期);第2页图1，"Text S1 Genotyping-by-sequencing protocol for PstI-MspI" * |
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