CN112921076A - Pig 50K liquid phase chip based on target capture sequencing and application thereof - Google Patents
Pig 50K liquid phase chip based on target capture sequencing and application thereof Download PDFInfo
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Abstract
The invention relates to a pig 50K liquid phase chip based on targeted capture sequencing and application thereof, wherein the pig 50K liquid phase chip is composed of an independently packaged pig 50K probe mixed solution and a hybridization capture reagent, and the pig 50K probe is a DNA double-strand probe and is a nucleotide sequence designed and synthesized according to a screened SNP locus; and the SNP locus is obtained by comparing the sequencing result of the whole genome of the pig to a pig reference genome for screening the SNP locus. The swine 50K liquid chip prepared by the method is mixed with a swine DNA high-throughput sequencing library, DNA fragments containing target sites in the swine DNA high-throughput sequencing library are captured for amplification and purification, and after high-throughput sequencing is carried out on products, sequencing results are returned to a swine reference genome for comparison, so that the genomic genotyping of the swine to be detected is obtained. The 50K liquid chip prepared by the invention is used for genotyping the pigs, and can solve the problems of high cost, inflexibility and incapability of large-scale use in pig farms in China in the prior art.
Description
Technical Field
The invention relates to the technical field of Genotyping, in particular to a pig 50K liquid chip based on a targeting capture Sequencing Genotyping (GBTS) technology and application thereof in Genotyping a pig genome.
Background
The Single Nucleotide Polymorphism (SNP) has the characteristics of large quantity, wide distribution, easy quick large-scale screening, convenient genotyping and the like, is a third-generation genetic marker following a polymorphism marker with the length of a first-generation restriction fragment and a second-generation microsatellite, namely a simple tandem repeat marker, is considered as the current best marker selection, and has important biological significance. At present, SNP markers become important tools for biological population identification, genetic structure analysis, functional gene mapping and genome selection. With the development of high-throughput SNP genotyping technology, methods based on whole genome or simplified genome sequencing have become the mainstream of SNP genotyping technology. For species with reference genome sequences, researchers generally design SNP chips by using known sequences, and can acquire SNP genotyping of corresponding sites by hybridizing randomly interrupted genomic DNA fragments with oligonucleotide probes on the chips.
At present, commercial SNP chips matured on pigs are mainly based on Illumina platform and Affymetrix platform. The former includes mainly PortineSNP 60 (containing 61565SNP), GGP-PortineHD (68528SNP, 80K for short), GGP-Portinesecond edition (containing 51000SNP, 50K for short), Compton's complex porcinNP 55 (containing 55000SNP), and Jiangxi university central core No. 1 (containing 51315 SNP). The Affymetrix platform mainly comprises a high-density chip developed by Affymetrix company, contains about 650000SNP, is high in price and is low in use amount. The two chips of the New York corporation (NeoGen) are mainly used in the current market in the largest amount, but for the practical application of molecular breeding, the price is still higher, the markers cannot be adjusted according to the self population, the flexibility is lacked, and the large-scale use in the pig farm in China is limited. Therefore, a new genotyping technology is urgently needed to reduce the manufacturing cost of high-throughput SNP chips and flexibly adjust SNP markers according to the actual population situation.
The target capture sequencing Genotyping (GBTS) is a technology for realizing deep resequencing of only a target site by reducing the abundance of a library, can obviously reduce the genotyping cost, and is not applied to pigs at present. In response, a pig high-throughput SNP probe is designed by using a targeted capture sequencing technology, and the GBTS technology is popularized to pig basic research and molecular breeding application in a large scale. Based on GBTS technology, we developed a brand-new pig 50K chip, called liquid chip, which has the following advantages, besides retaining the advantages of high detection accuracy and low price, compared with the chips developed based on the illumina platform and Affymetrix platform (hereinafter referred to as solid chip) in the market mainstream: the method has the advantages that the method is completely free of domestic independent intellectual property rights and political trade risks; the density of the developed SNP chip can be flexibly adjusted, and can be reduced to 1K with low density or expanded to 200K with high density on the basis of the mainstream 50K; new sites can be supplemented at any time, a small amount of samples can be detected, and the sample size is not limited; the detection cost is lower than that of the current solid phase chip.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a pig 50K liquid chip based on targeted capture sequencing and application thereof, and simultaneously provides a special SNP probe design principle and a pig whole genome 50K liquid chip.
The invention provides a pig 50K liquid phase chip based on targeted capture sequencing, which is composed of an independently packaged pig 50K probe mixed solution and a hybridization capture reagent, wherein the pig 50K probe is a DNA double-strand probe and is a nucleotide sequence designed and synthesized according to a screened SNP locus; the SNP locus is obtained by comparing the sequencing result of the whole genome of the pig to the reference genome of the pig to screen the SNP locus, and the screening principle is as follows: the distribution of each chromosome is uniform, the distribution of both ends of each chromosome is dense, and the site polymorphism is good, wherein the index of the site polymorphism is that MAF is more than 0.35 in Duroc, Changbai pigs and big white pigs; the coverable area of the screened SNP locus simultaneously meets the design principle of a pig 50K probe; the design principle of the pig 50K probe is as follows: the length of the probe is 110bp, the GC content of the probe is 30-80%, the number of homologous regions is less than or equal to 5, and the selected region does not contain SSR and GAP regions to the maximum extent; designing two nucleotide sequences which are overlapped by 60-70% and cover the SNP locus according to the SNP locus obtained by screening; synthesizing single-stranded nucleotide according to the designed nucleotide sequence, wherein the two synthesized DNA nucleotide sequences with the length of 110bp and the 5' end modified by a biotin group are called as pig 50K probes; and mixing the two synthesized pig 50K probes by equal molar mass, and using EDTA and Tris-HCl mixed liquor to fix the volume of the pig 50K probe mixed liquor to 3 pmol/mL.
Preferably, the hybrid capture reagent is a GenoBaits DNA-seq Library Prep kit from borudidi biotechnology limited, comprising GenoBaits Block I, GenoBaits Block II, GenoBaits 2 × Hyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2 × Beads Wash Buffer, GenoBaits 10 × Wash Buffer I, GenoBaits 10 × Wash Buffer II, GenoBaits 10 × Wash Buffer III, GenoBaits 10 × strand Wash Buffer II, and GenoBaits 10 × strand Wash Buffer in separate packages.
Preferably, the SSR region is a simple repeated sequence with a core sequence of 1-6 bp.
Preferably, the porcine reference genome is version 11.1.
The invention provides a pig genome genotyping method based on targeted capture sequencing, which comprises the following steps:
s1, preparing the pig 50K liquid phase chip based on the target capture sequencing;
s2, constructing a pig DNA high-throughput sequencing library by using the genome DNA of the pig to be detected;
s3, mixing the swine 50K liquid chip prepared in the S1 with the swine DNA high-throughput sequencing library constructed in the S2, and capturing DNA fragments containing target sites in the swine DNA high-throughput sequencing library;
and S4, amplifying and purifying the DNA fragment obtained in the S3, and after high-throughput sequencing of the product, replying the sequencing result to a pig reference genome for comparison so as to obtain the genome genotyping of the pig to be detected.
Further, the method for constructing the pig DNA high-throughput sequencing library of S2 comprises the following steps: the method comprises the steps of fragmenting the genome DNA of a pig to be detected by adopting an ultrasonic wave breaking method, obtaining a pig DNA high-throughput sequencing library through the steps of filling in and adding A tail at the tail end, connecting a sequencing joint and the like, and detecting the concentration of the pig DNA high-throughput sequencing library by utilizing the Qubit.
Further, the capturing method of S3 is: mixing a pig 50K probe containing biotin and magnetic beads covered by streptavidin, adsorbing target pig genome DNA fragments onto the magnetic beads through the combination of the biotin and the streptavidin, and then washing away DNA fragments in non-target areas through an elution process, thereby obtaining the required pig genome DNA fragments.
Further, in step S4, the high throughput sequencing data is processed by BWA and GATK software to obtain SNP genotyping of individual specific sites.
The swine 50K liquid chip provided by the invention is applied to swine molecular breeding.
Compared with the prior art, the pig 50K liquid chip based on the target capture sequencing and the pig genome genotyping method have the following beneficial effects:
1. the invention provides a pig high-throughput SNP50K probe for genotyping based on targeted capture sequencing, which considers the distribution condition of captured SNP sites in the whole genome during probe design, considers the problem of site polymorphism in capture probes, requires the MAF to be more than 0.35 in Duroc, long white pigs and large white pig groups, and effectively avoids the problems of uneven labeling density and poor polymorphism possibly caused by simplifying the genome sequencing and typing technology.
2. The invention designs 2 probes with GC content controlled between 30-80% and number of homologous regions less than or equal to 5 for 80bp before and after each SNP marker locus, effectively improves the hybridization stability of each SNP sequence and improves the efficiency of fragment capture. Meanwhile, the specific specificity of the site flanking sequences on the genome is fully considered, the site specific capture rate is ensured, and the sequencing cost is reduced. The invention theoretically can design a capture probe for any specific SNP locus, so the invention has important application in large-scale SNP typing of specific markers of pigs, and provides an efficient genome typing technology for genetic diversity analysis, strain identification, marker-assisted breeding research and application of pigs.
3. The swine 50K liquid phase chip can supplement SNP sites at any time, and a new probe is designed according to the new sites and added into the existing swine 50K probe mixed liquid. The present invention can also remove unwanted SNP markers without performing sequencing assays. In the prior art, the marking sites of the solid phase chip are fixed, and the increase and decrease of the SNP sites can be realized only by designing a new chip. In addition, the solid phase chip technology has requirements on the sample amount, one chip can be used for preparing 12 samples, 24 samples, 48 samples or even 96 samples, the requirement on the sample conveying amount is met, the whole experiment is reacted in a PCR tube, a reaction system can be added according to the requirement, 1 sample can be detected, the sample detection amount is flexible, the technical defects of the solid phase chip are made up, and the method has higher popularization value in pig molecular breeding.
4. By optimizing the capture hybridization system, the DNA hybridization capture time of the step S3 is 1 hour, compared with the overnight hybridization capture process of more than 16 hours, the genotype acquisition time can be greatly shortened, the whole process of library construction and capture can be completed in one day, and the time is saved for genotype detection.
5. The probe has better tolerance to flanking sequences, so the swine 50K SNP liquid-phase chip has higher tolerance to the variation condition of the flanking sequences, and can still stably capture a target sequence under the condition that the variation of the flanking sequences is not higher than 10 percent, thereby not only obtaining the target SNP information, but also obtaining the sequence information of 80bp of each upstream and downstream, including SNP variation information of more than 200K, and providing more information support for the molecular breeding of swine under the condition of the same cost.
Drawings
FIG. 1 is a schematic diagram of designing a probe according to SNP sites according to the present invention.
FIG. 2 is a diagram showing the distribution of SNP sites on different chromosomes in a liquid chip for swine 50K according to the present invention;
FIG. 3 is a diagram showing the interval between adjacent SNP sites of a liquid phase chip of 50K for a pig provided by the present invention;
FIG. 4 is a PCA diagram of the swine 50K liquid phase chip provided by the invention for distinguishing three swine breeds.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 pig 50K liquid chip based on targeted capture sequencing and method for applying same to pig genome genotyping
As shown in figure 1, a plurality of probes can be designed according to the screened SNP sites, and the probe sequence determines the accuracy and the sensitivity of the swine 50K liquid phase chip.
S1, preparing a pig 50K liquid phase chip based on targeted capture sequencing, wherein the pig 50K liquid phase chip is composed of an independently packaged pig 50K probe mixed solution and a hybridization capture reagent, and the pig 50K probe is a DNA double-strand probe and is a nucleotide sequence designed and synthesized according to the screened SNP sites; the SNP locus is obtained by comparing the sequencing result of the whole genome of the pig to the reference genome of the pig to screen the SNP locus, and the screening principle is as follows: the distribution of each chromosome is uniform, the distribution of both ends of each chromosome is dense, and the site polymorphism is good, wherein the index of the site polymorphism is that MAF is more than 0.35 in Duroc, Changbai pigs and big white pigs; the coverable area of the screened SNP locus simultaneously meets the design principle of a pig 50K probe; the design principle of the pig 50K probe is as follows: the length of the probe is 110bp, the GC content of the probe is 30-80%, the number of homologous regions is less than or equal to 5, and the selected region does not contain SSR and GAP regions to the maximum extent; designing two nucleotide sequences which are overlapped by 60-70% and cover the SNP locus according to the SNP locus obtained by screening; synthesizing single-stranded nucleotide according to the designed nucleotide sequence, wherein the two synthesized DNA nucleotide sequences with the length of 110bp and the 5' end modified by a biotin group are called as pig 50K probes; and mixing the two synthesized pig 50K probes by equal molar mass, and using EDTA and Tris-HCl mixed liquor to fix the volume of the pig 50K probe mixed liquor to 3 pmol/mL.
In this example, the hybrid capture reagent is a GenoBaits DNA-seq Library Prep kit from borudidi biotechnology limited, including GenoBaits Block I, GenoBaits Block II, GenoBaits 2 × Hyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2 × Beads Wash Buffer, GenoBaits 10 × Wash Buffer I, GenoBaits 10X Wash Buffer II, GenoBaits 10X Wash Buffer III, GenoBaits 10X strand Wash Buffer.
In this embodiment, the SSR region is a simple repeat sequence having a core sequence of 1-6 bp.
In this example, the porcine reference genome is version 11.1.
In this example, 55048 SNP sites are co-targeted to design a pig 50K probe, and the obtained SNP covered by a pig 50K liquid phase chip is shown in FIGS. 2-3, and FIG. 2 shows that each chromosome is covered by the SNP marker screened by the pig 50K liquid phase chip of the present invention, and the design is basically carried out according to the chip design principle; FIG. 3 illustrates that the SNP intervals of the 50K liquid phase chip of the present invention are reasonable, averaging about 40KB, and are approximate for each chromosome.
S2, constructing a pig DNA high-throughput sequencing library by using the genome DNA of the pig to be detected;
s2.1, extracting the genome DNA of the pig to be detected:
20 samples of each of three pig varieties of Duroc, Changbai and Dabai are selected, and the ear tissues of 60 samples are totally extracted for genome DNA. The specific method comprises the following steps:
(1) cutting appropriate amount of ethanol dehydrated pig ear tissue, placing into 96 deep-well plate (2.0 mL centrifuge tube can be used if the amount is small), adding 1 steel ball of 5mm, freezing with liquid nitrogen, and grinding for 1-2min with grinder.
(2) 500uL Buffer PL2 and 5uL protease K of 20mg/mL were added to the deep well plate, the cover was closed, and the mixture was shaken and mixed by a shaker.
(3) Putting into 65 deg.C water bath for 30min, taking out from the middle of the water bath, and mixing.
(4) Centrifuge for 10min at 4000rpm, transfer 400uL of supernatant to a new 96-well deep-well plate. (care must not be taken to suck in intermediate precipitates).
(6) Adding 800uL of PW solution, and fully and uniformly mixing.
(7) Transferring the supernatant obtained in the step 6 into a 96-hole centrifugal column in two times, and performing vacuum filtration.
(8) Adding 600uL WBI into a 96-hole centrifugal column, standing at room temperature for 2min, and carrying out vacuum filtration. (confirmation that absolute ethanol was added to WB I in the volume specified on the bottle).
(9) 600uL WBII was added to a 96-well spin column and vacuum filtered. (confirmation that absolute ethanol was added to WB II in the volume specified on the bottle).
(10) 600uL WBII was added to a 96-well spin column and vacuum filtered.
(11) The 96-well spin column was placed in an empty collection plate and centrifuged at 4000rpm for 5 min. The 96-well spin column was placed on a new 96-well PCR plate and air dried at room temperature.
(12) Adding 60-100 uL of 65 ℃ preheating TE into a 96-hole centrifugal column, standing at room temperature for 2min, centrifuging at 4000rpm for 5min, and throwing the DNA solution into a 96-hole PCR plate. Preheating TE to 65 ℃ is beneficial to improving the elution efficiency of DNA and detecting the length of a target fragment by sugar gel electrophoresis.
S2.2, constructing a pig DNA high-throughput sequencing library
S2.2.1 the following reaction system was prepared in a total volume of 20ml in a PCR tube: 200 ng of pig DNA obtained in the step 1, 4 mu L of GenoBaits End Repair Buffer, 3.1 mu L of GenoBaits End Repair enzyme and the balance of ultrapure water; the reagents used in this example were all from the GenoBaits DNA-seq Library Prep kit from Shijiazhuang Boridi Biotech, Inc.
S2.2.2 the reaction system was gently mixed and centrifuged briefly to collect the reaction solution at the bottom of the tube.
S2.2.3 the reaction tube was placed in a PCR instrument to perform the following reactions, 82 ℃ hot lid:
TABLE 1
| 37℃ | 20min |
| 72℃ | 20min |
| HoLd at | 4℃ |
S2.2.4 the following components were added directly to the above reaction tube: the total amount is 20 μ L, wherein the total amount is 2 μ L of GenoBaits ULtra DNA Ligase, 8 μ L of GenoBaits ULtra DNA Ligase Buffer, 2 μ L of GenoBaits Adapter for MGI, and the balance ultrapure water;
s2.2.5 the reaction system was gently mixed and centrifuged briefly to collect the reaction solution at the bottom of the tube. The reaction system must be fully mixed, otherwise the library building failure can be caused.
S2.2.6 the reaction tube was placed in a PCR instrument and the following reactions were performed, with the hot lid removed:
TABLE 2
| 22℃ | 60min |
| HoLd at | 4℃ |
S2.2.7 mu.L of DNA purified magnetic beads which have been equilibrated at room temperature for more than 30 minutes are added to the above system, mixed by shaking to make bubbles as little as possible, left to stand for 5 minutes, and then centrifuged briefly to collect the liquid at the bottom of the tube.
S2.2.8 was placed on a magnetic rack for at least 3min until the solution was clear and the supernatant was removed.
S2.2.9 the PCR tubes were kept in a magnetic rack and 100. mu.L of 80% ethanol was added. Incubate at room temperature for 30 seconds and remove the supernatant.
S2.2.10 the PCR tubes were kept in a magnetic rack and left uncovered and allowed to air for 5 minutes until the ethanol had evaporated to dryness.
S2.2.11 the PCR tube was removed from the magnetic rack and the beads were allowed to air dry.
The above 2.1-2.12 steps are completed in one day.
S2.2.12 the following reaction system was prepared in a new PCR tube: the total amount was 20. mu.L, of which GenoBaits PCR Master Mix 10. mu. L, I5 Barcode (10 μm) -MGI 1. mu. L, I7 Barcode (2 μm) -MGI 5. mu.L, and 4. mu.L of ultrapure water.
S2.2.13 the system was added to a 2.12 PCR tube and the air-dried beads were resuspended and the reaction was collected to the bottom of the tube by brief centrifugation.
S2.2.14 the reaction tube was placed in a PCR machine to perform the following reactions:
TABLE 3
The number of amplification cycles is adjusted based on the initial amount of DNA, which is:
TABLE 4
S2.2.15 adding 20 μ L DNA purified magnetic beads which have been balanced at room temperature for more than 30min into the system, shaking and mixing uniformly, avoiding generating air bubbles as much as possible, standing for 5min, centrifuging for a short time, and collecting the liquid to the bottom of the tube;
s2.2.16 placing the PCR tube on a magnetic frame for at least 3min until the solution is clear, and removing the supernatant;
s2.2.17 the PCR tubes were kept in a magnetic rack and 100. mu.L of 80% ethanol was added. Incubation at room temperature for 30 seconds, removing the supernatant;
s2.2.18 the PCR tubes were kept in a magnetic rack and left to air open for 10 minutes.
S2.2.19 taking out the PCR tube from the magnetic frame, adding 35 μ L Tris-HcL, shaking, mixing, standing for 5min, centrifuging for a short time, and collecting the liquid to the bottom of the tube.
S2.2.21 the PCR tube is placed on a magnetic rack and when the solution is clear (about 3 minutes), the supernatant is transferred to a new tube to complete the construction of the DNA sequencing library.
S2.2.22 library quality testing: taking 1 microliter of the prepared library sample, and quantifying the library sample by using the Qubit, wherein the total amount of DNA of a sequencing library is required to be more than 500 ng; taking 3 μ L of the prepared library sample, performing a 1% agarose gel electrophoresis experiment, wherein the range of the electrophoresis result DNA fragment is 300-500bp, so as to ensure the effect of the subsequent capture experiment. The prepared library sample can be stored at 4 ℃ for a short time and at-20 ℃ for a long time.
S3, mixing the swine 50K liquid chip prepared in the S1 with the swine DNA high-throughput sequencing library constructed in the S2, and capturing DNA fragments containing target sites in the swine DNA high-throughput sequencing library;
s3.1, balancing the DNA purification magnetic beads for more than 30 minutes at room temperature, uniformly mixing the pig 50K probe and the DNA purification magnetic beads in a 0.2mL PCR tube, and centrifuging for a short time;
s3.2 the following reagents were mixed in the PCR tube:
500 ng/library of the pig DNA high-throughput sequencing library, 5 mu g (5 mu L) of GenoBaits BLOCK I, 2 mu L of GenoBaits BLOCK II for ILM/MGI and 300ng of the probe form a liquid phase chip mixed solution containing the pig DNA high-throughput library and the pig 50K probe;
s3.3, concentrating the PCR tube to be completely dry at the temperature of less than or equal to 60 ℃ by using a vacuum concentrator;
s3.4 after the concentration is finished, the PCR tube needs to be centrifuged at 12000rpm for 1min and then subjected to subsequent operation, and the step can be suspended and stored overnight at room temperature (15-25 ℃);
s3.5 dissolving all GenoBaits hybridization reagents at room temperature; if the GenoBaits 2X Hyb Buffer is crystallized, heating to 65 ℃, shaking once at intervals until the crystals are completely dissolved;
s3.6 the following reagent system was added to S3.3.4 completed PCR tubes:
TABLE 5
| 8.0 |
| 2.0μL GenoBaits Hyb Buffer Enhancer V3.3 |
| 6.0μL NucLease-Free Water |
S3.7, sucking or vortex mixing the system in the PCR tube, centrifuging at 12000rpm for 1min, standing at room temperature for 5min, sucking or vortex mixing again, slightly centrifuging, and transferring all the mixed solution in the PCR tube into a 0.2mLEP tube;
s3.8 incubating the LEP tube under the condition of 95 ℃ of a PCR amplification instrument for 10min in a thermal cycle (the temperature of a thermal cover is 105 ℃);
s3.9 when the temperature of the PCR amplification instrument is reduced to 65 ℃, the LEP tube is transferred to another 65 ℃ PCR instrument with a hot cover of 75 ℃, and the temperature is bathed for 1 hour.
S3.10 the eluate requiring preheating at 65 ℃ is placed in the PCR apparatus in advance, and the rest of the eluate is stored at room temperature. The eluent was prepared in advance as follows:
TABLE 6
| Buffer | Water (W) | Total amount of | |
| |
250μL | | 500μL |
| GenoBaits | |||
| 10×Wash Buffer I | 28μL | | 280μL |
| GenoBaits | |||
| 10×Wash Buffer Ⅱ | 18μL | | 180μL |
| GenoBaits | |||
| 10×Wash Buffer Ⅲ | 18μL | | 180μL |
| GenoBaits | |||
| 10×Stringent Wash Buffer | 40μL | | 400μL |
| GenoBaits | |||
| 2×Beads Wash Buffer | 250μL | 250μL | 500μL |
S3.10.1 Single Capture System, Dilute GenoBaits buffers to a 1 × System. The 1 × system can be stored at room temperature for 4 weeks.
TABLE 7
| Buffer | Water (W) | Total amount of | |
| |
250μL | | 500μL |
| GenoBaits | |||
| 10×Wash Buffer I | 28μL | | 280μL |
| GenoBaits | |||
| 10×Wash Buffer Ⅱ | 18μL | | 180μL |
| GenoBaits | |||
| 10×Wash Buffer Ⅲ | 18μL | | 180μL |
| GenoBaits | |||
| 10×Stringent Wash Buffer | 40μL | | 400μL |
| GenoBaits | |||
| 2×Beads Wash Buffer | 250μL | 250μL | 500μL |
The 10 xWash Buffer I and the 10 xS-W Buffer can precipitate at normal temperature, and the precipitate can be dissolved at 65 ℃.
S3.10.2 Wash Buffer I and Stringent Wash Buffer storage
TABLE 8
S3.11 preparing DNA purification magnetic beads, wherein the DNA purification magnetic beads are prepared immediately before use, namely the preparation is carried out immediately, and the preparation steps are as follows: placing the DNA purified magnetic beads at room temperature for 10min before use, and uniformly mixing by vortex oscillation for 15 s;
S3.12A reaction preparation 50. mu.L of DNA purification magnetic beads were placed in a 0.2mL EP tube;
s3.13 the EP tube is placed on a magnetic frame to completely separate the magnetic beads from the solution. The supernatant was removed and the beads were retained.
S3.14 to the above EP tube, 150. mu.L of GenoBaits 1X Bead Wash Buffer was added, vortexed for 10S, the EP tube was transferred to a magnetic rack, the magnetic beads were completely separated from the solution, and the supernatant was removed.
S3.15 repeat the above step 6 twice, 3 total washes.
S3.16 transfer 16. mu.L of the hybridization solution incubated at 65 ℃ for 1 hour to a prepared EP tube obtained at 3.15, operate on a PCR instrument, vortex for 10S, mix well, and centrifuge for 5S.
S3.17 put the EP tube into a PCR instrument, at 65 ℃, 45min, and 75 ℃ of a hot cover, to combine the DNA with the magnetic beads, and shake for 5S every 12 min.
S3.18 Add 100. mu.L 65 ℃ preheated GenoBaits 1 × Wash Buffer I to each EP tube, vortex for 5S briefly, centrifuge for 5S, then place on magnetic stand, completely separate magnetic beads from solution, remove supernatant (unbound DNA);
s3.19 Add 150. mu.L of a preheated (65 ℃) GenoBaits 1X Stringent Wash Buffer, and pipette up and down slowly 10 times (avoiding the formation of bubbles); after the last group of samples are mixed uniformly, placing for 2min (the time requirement is accurate); placing on a magnetic frame, completely separating the magnetic beads from the solution, and quickly removing the supernatant by using a liquid transfer device.
S3.20 repeat step 3.19
S3.21, adding 150 mu L of room-temperature GenoBaits 1X Wash Buffer I, shaking for 2min, placing on a magnetic frame, completely separating magnetic beads from the solution, and removing supernatant;
s3.22, adding 150 mu L of GenoBaits 1X Wash Buffer II at room temperature, shaking for 1min, placing on a magnetic rack, completely separating magnetic beads from the solution, and removing supernatant;
s3.23, adding 150 mu L of GenoBaits 1X Wash Buffer III at room temperature, shaking for 30S, placing on a magnetic rack, completely separating magnetic beads from the solution, and removing supernatant;
s3.24 remove the tubes containing the captured DNA beads from the magnetic rack
S3.25 adding 20 u L NucLease-Free Water, suction and beating 10 times, ensuring all the magnetic beads heavy suspension, taking care not to discard the magnetic beads, using 10 u L magnetic beads heavy suspension for the following PCR amplification and purification, the remaining 10 u L for backup.
And S4, amplifying and purifying the DNA fragment obtained in the S3, and after high-throughput sequencing of the product, replying the sequencing result to a pig reference genome for comparison so as to obtain the genome genotyping of the pig to be detected.
S4.1 according to the library type, PCR reagents were prepared in 0.2mL PCR tubes:
GenoBaits PCR Master Mix 15. mu.L, GenoBaits Primer Mix for MGI 1.2. mu.L, DNA-captured magnetic beads 10. mu.L, and ultrapure water 3.8. mu.L, in a total amount of 30. mu.L.
S4.2, briefly vortexing and centrifuging to ensure that the magnetic beads are still in the solution;
s4.3, putting the PCR tube into a PCR instrument, carrying out PCR amplification according to the following procedures at the temperature of a hot cover of 105 ℃:
TABLE 9
The PCR amplification product can be stored overnight at 4 ℃.
S4.4, adding 45 mu L (1.5X voLume) of magnetic beads with captured DNA into each PCR reaction, shaking and uniformly mixing the mixture to avoid generating bubbles as much as possible, standing the mixture for 5min, and collecting the liquid to the bottom of a tube by short-time centrifugation; placing on a magnetic frame for at least 3min until the solution is clear, and removing the supernatant;
s4.5, keeping the PCR tube in the magnetic frame, adding 100 mu L of 80% ethanol, incubating at room temperature for 30 seconds, removing the supernatant, keeping the PCR tube in the magnetic frame, uncovering and airing for 10 minutes;
s4.6, taking out the PCR tube from the magnetic frame, adding 35 mu L of Tris-HcL, shaking, uniformly mixing, standing for 5min, and centrifuging for a short time to collect liquid to the bottom of the tube; on the magnetic rack, when the solution is clear (about 3 minutes), the supernatant is transferred to a new tube for-20 ℃ storage, and the purified PCR fragment can be stored at-20 ℃ for one week.
S4.7, measuring the library by using a Qubits FLuorometer and a Qubit dsDNA HS Assay Kit, and measuring the average length of the fragments of the captured DNA library on a digital electrophoresis system; library concentrations were measured with the KAPA Library Quantification Kit.
S4.8 Probe sequencing was performed using a sequencer.
Example 2 genotyping quality evaluation of the porcine 50K liquid chip obtained in example 1
Three major pig breeds, namely, Duroc, Changbai and Dabai, which are the largest in feeding quantity at present in China, are randomly selected for 20 heads respectively, the 50K liquid chip of the pig obtained in the embodiment 1 is adopted for genotyping detection, and the following quality evaluation is carried out on the genotype.
1. Detection rate
The SNP detection rate and the individual detection rate are important indexes for measuring the quality of the chip, and are generally measured by the sites of an autosome and an X chromosome. The detection rate of SNP of the swine 50K liquid phase chip of the embodiment 1 is high, the average detection rate of SNP is 99.88%, and the standard deviation is 0.007. 60 individuals have SNP detection rates of more than 99 percent, the maximum value and the minimum value of the individual detection rates are 99.92 percent and 99.19 percent respectively, the average detection rate is 99.76 percent, and the standard deviation is 0.0016; thus, the genotype of the pig 50K liquid phase chip of example 1 was examined with good quality.
TABLE 1060 head Duroc, Changbai and Dabai pigs 50K liquid phase core SNP detection rates
| Standard of merit | Ratio of | Average detection rate | Standard deviation of | |
| Duroc | >85％ | 99.53％ | 99.85％ | 0.009 |
| Changbai (white and long) | >90％ | 99.34％ | 99.88％ | 0.007 |
| White spirit | >95％ | 98.82％ | 99.93％ | 0.004 |
2. Stability of genotyping
Stability is generally measured by the consistency and correlation coefficient of the results of two genotype tests on duplicate samples. The results of two genotype tests on 6 replicates using 6 duroc, Changbai and Dabai pigs to test the 50K liquid phase chip from the pig of example 1 are as follows: the genotype consistency is 99.3%, and the correlation coefficient is 99.7%, which shows that the genotype detection stability of the swine 50K liquid phase chip of example 1 is good.
3. Analysis of genetic Structure
Whether SNP markers can be used for genetic structure division, FIG. 4 shows the genotype of 60 Duroc, Changbai and Dabai determined using the swine 50K SNP liquid phase chip of example 1, and three varieties were divided by Principal Component Analysis (PCA). As shown in FIG. 4, the swine 50K SNP liquid-phase chip of example 1 can clearly distinguish three breeds.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A pig 50K liquid phase chip based on targeted capture sequencing is characterized in that the pig 50K liquid phase chip is composed of an independently packaged pig 50K probe mixed solution and a hybridization capture reagent, wherein the pig 50K probe is a DNA double-strand probe and is a nucleotide sequence designed and synthesized according to a screened SNP locus; the SNP locus is obtained by comparing the sequencing result of the whole genome of the pig to the reference genome of the pig to screen the SNP locus, and the screening principle is as follows: the distribution of each chromosome is uniform, the distribution of both ends of each chromosome is dense, and the site polymorphism is good, wherein the index of the site polymorphism is that MAF is more than 0.35 in Duroc, Changbai pigs and big white pigs; the coverable area of the screened SNP locus simultaneously meets the design principle of a pig 50K probe; the design principle of the pig 50K probe is as follows: the length of the probe is 110bp, the GC content of the probe is 30-80%, the number of homologous regions is less than or equal to 5, and the selected region does not contain SSR and GAP regions to the maximum extent; designing two nucleotide sequences which are overlapped by 60-70% and cover the SNP locus according to the SNP locus obtained by screening; synthesizing single-stranded nucleotide according to the designed nucleotide sequence, wherein the two synthesized DNA nucleotide sequences with the length of 110bp and the 5' end modified by a biotin group are called as pig 50K probes; and mixing the two synthesized pig 50K probes by equal molar mass, and using EDTA and Tris-HCl mixed liquor to fix the volume of the pig 50K probe mixed liquor to 3 pmol/mL.
2. The pig 50K liquid phase chip based on targeted capture sequencing according to claim 1, wherein the hybridization capture reagent is a GenoBaits DNA-seq Library Prep kit from Boyle biotechnology, Inc., Shichaga, and comprises GenoBaits Block I, GenoBaits Block II, GenoBaits 2 XHyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2 XBeads Wash Buffer, GenoBaits 10 XWash Buffer I, GenoBaits 10 XWash Buffer II, GenoBaits 10 XWaits 10 XWash Buffer III, GenoBaits 10X Stringent Wash Buffer in independent packages.
3. The pig 50K liquid phase chip based on the targeted capture sequencing of claim 1, wherein the SSR region is a simple repeat sequence with a core sequence of 1-6 bp.
4. The pig 50K liquid phase chip based on the target capture sequencing of claim 1, wherein the pig reference genome is version 11.1.
5. A pig genome genotyping method based on targeted capture sequencing is characterized by comprising the following steps:
s1, preparing the pig 50K liquid phase chip based on the target capture sequencing of claim 1;
s2, constructing a pig DNA high-throughput sequencing library by using the genome DNA of the pig to be detected;
s3, mixing the swine 50K liquid chip prepared in the S1 with the swine DNA high-throughput sequencing library constructed in the S2, and capturing DNA fragments containing target sites in the swine DNA high-throughput sequencing library;
and S4, amplifying and purifying the DNA fragment obtained in the S3, and after high-throughput sequencing of the product, replying the sequencing result to a pig reference genome for comparison so as to obtain the genome genotyping of the pig to be detected.
6. The method for genotyping pig genomes based on targeted capture sequencing according to claim 5, wherein the method for constructing the pig DNA high-throughput sequencing library of S2 is as follows: the method comprises the steps of fragmenting the genome DNA of a pig to be detected by adopting an ultrasonic wave breaking method, obtaining a pig DNA high-throughput sequencing library through the steps of filling in and adding A tail at the tail end, connecting a sequencing joint and the like, and detecting the concentration of the pig DNA high-throughput sequencing library by utilizing the Qubit.
7. The method for genotyping pig genomes based on targeted capture sequencing according to claim 5, wherein the capture method of S3 is: mixing a pig 50K probe containing biotin and magnetic beads covered by streptavidin, adsorbing target pig genome DNA fragments onto the magnetic beads through the combination of the biotin and the streptavidin, and then washing away DNA fragments in non-target areas through an elution process, thereby obtaining the required pig genome DNA fragments.
8. The method for genotyping pig genomes based on targeted capture sequencing according to claim 5, wherein in S4, the SNP genotyping of individual specific sites is obtained after the high-throughput sequencing data is processed by BWA and GATK software.
9. The use of the porcine 50K liquid-phase chip of claim 1 in molecular breeding of swine.
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