CN117363750A - Preparation method of 15K liquid phase chip for Min pig breeding and genotyping method - Google Patents

Preparation method of 15K liquid phase chip for Min pig breeding and genotyping method Download PDF

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CN117363750A
CN117363750A CN202311488548.0A CN202311488548A CN117363750A CN 117363750 A CN117363750 A CN 117363750A CN 202311488548 A CN202311488548 A CN 202311488548A CN 117363750 A CN117363750 A CN 117363750A
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min pig
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张冬杰
刘娣
何鑫淼
王文涛
田明
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Institute of Animal Husbandry of Heilongjiang Academy of Agricultural Sciences
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Abstract

The invention discloses a preparation method of a 15K liquid phase chip for breeding Min pigs and a genotyping method, which aim to solve the problem that a chip for breeding and a solid phase chip for breeding are not flexible at present in the absence of a local pig. The Min pig 15K liquid phase chip is composed of a separately packaged Min pig 15K probe mixed solution and a hybridization capture reagent, wherein the Min pig 15K probe is a nucleotide sequence designed and synthesized for SNP loci screened by Min pig whole genome sequences. The Min pig 15K liquid phase chip prepared by the method is mixed with a Min pig DNA high-throughput sequencing library to be detected, DNA fragments containing target sites in the Min pig DNA high-throughput sequencing library to be detected are captured for amplification and purification, and after high-throughput sequencing, sequencing results are returned to a pig reference genome for comparison, so that genome genotyping of the Min pig to be detected is obtained. The invention can be used for the breeding and the breeding work of the Min pigs, and solves the problem that the chip special for breeding local pig breeds is lacking in China at present.

Description

Preparation method of 15K liquid phase chip for Min pig breeding and genotyping method
Technical Field
The invention belongs to the technical field of biological breeding, and particularly relates to a preparation method and a genotyping method of a 15K liquid phase chip for Min pig breeding.
Background
The breeding industry is in vibration and the fine breed is in advance, and the fine breed pig group is the premise and the foundation of the development of the modern live pig industry and has very key effect in promoting the yield increase and the effect improvement. The genetic resource of local pigs in China is valuable wealth from five thousand years of farming civilization, is a fresh and alive carrier for carrying the Chinese civilization, and is an important strategic resource for maintaining national ecological safety and agricultural safety. The method has the advantages of protecting and utilizing the genetic resources of the square pigs well, and has great significance for improving the comprehensive production capacity and the core competitiveness of the animal husbandry in China, deeply implementing the strategy of village plain vibration and better meeting the requirements of increasingly growing, continuously upgrading and personalizing animal products of vast people.
Due to the presence of genetic variation, different varieties or populations exhibit phenotypic differences. Genetic variation is mainly reflected by genetic markers, and genetic markers which are widely used at present are SNP markers at the molecular level. The main detection methods of SNP markers are a chip detection method and a high throughput sequencing method. The chip is used for SNP detection, and has the characteristics of simple operation, high flux and the like, and currently, the chip mainly comprises a solid-phase chip and a liquid-phase chip. On pigs, there are already mature commercial chips such as 60K chip (Porcine SNP 60), core one chip, etc., which are all solid phase chips. In use, people gradually find that the solid-phase chip has some defects, such as (1) fixed detection sites, inflexibility and slow updating. (2) The number of marker loci is relatively small compared to the entire genome, and a large number of genomic genetic variations are not detected. (3) The detection site has group specificity, taking a 60K chip of a pig as an example, and the information of site design is derived from wild boars and some common introduced varieties (Duroc pigs, changbai pigs, dabai pigs and Pitch pigs), so that the chip is used for detecting other groups (such as some local varieties), and the applicability is possibly poor due to larger genetic differences among the groups. (4) Because the solid phase chip is designed based on the prior knowledge, the solid phase chip technology cannot detect the new mutation in the group. The liquid phase chip makes up the deficiency of the solid phase chip to a certain extent, the site selection is more flexible, the SNP quantity selection range is wider, and the specific detection chip can be designed for specific groups.
At present, the mature commercial chip on pigs still takes a solid-phase chip as a main component, but takes a liquid-phase chip taking a targeted capture sequencing technology as a main component, and various products such as a 'Jia Dou Yi' chip for detecting a family donkey genome, a 'Shu Chi 1' chip for breeding a yak genome, a 'Shennong 1' chip special for a first Chinese local chicken and the like are also obtained. Besides the advantages of high detection accuracy and lower price, the liquid phase chip has the advantages of independent intellectual property rights, flexible adjustment of developed SNP chip density, capability of supplementing new sites at any time, capability of being combined with other chips for use, capability of designing a special chip for a certain group, no sample size limitation and the like.
For example, CN 112921076A, entitled "preparation method of pig 50K liquid phase chip based on targeted capture sequencing and application thereof", the pig 50K liquid phase chip is composed of separately packaged pig 50K probe mixed solution and hybridization capture reagent, the pig 50K probe is DNA double-stranded probe, and is a nucleotide sequence designed and synthesized according to the screened SNP site; the SNP locus is obtained by comparing the whole genome sequencing result of the pig to the reference genome of the pig for screening the SNP locus. The pig 50K liquid phase chip prepared by the method is mixed with a pig DNA high-throughput sequencing library, DNA fragments containing target sites in the pig DNA high-throughput sequencing library are captured for amplification and purification, and after high-throughput sequencing, the products are returned to a pig reference genome by using a sequencing result for comparison, so that the genome genotype of the pig to be detected is obtained. The 50K liquid phase chip prepared by the method disclosed by the invention is used for genotyping pigs, so that the problems that the prior art is high in cost and inflexible and cannot be used in large scale in pig farms in China can be solved. The invention provides a pig high-throughput SNP50K probe for genotyping based on targeted capture sequencing, wherein the distribution of captured SNP loci in a whole genome is considered in the process of probe design, the problem of locus polymorphism is considered in the capture probes, MAF requirement in Duroc, changbai pig and Dabai pig groups is more than 0.35, and the problems of uneven marker density and poor polymorphism possibly caused by simplifying a genome sequencing typing technology are effectively avoided.
The 50K liquid phase chip of the pig is developed based on the introduced variety Duchesner growth design, is not suitable for local pig varieties in China, is suitable for Min pigs, and can be popularized to other local pigs. Meanwhile, compared with 50K liquid phase chips, the number of SNP loci selected in the patent is reduced by 35000, the use cost is greatly reduced, and the genotyping requirement can be met.
The Min pig is a typical representative of North China pig breeds, is the only national grade protection breed selected in northeast, and is the domestic pig breeds in northeast. It has excellent characteristics of excellent meat quality, high fertility, cold resistance, coarse feeding resistance and the like, but has the disadvantages of low growth speed, low lean meat percentage and the like. In the eighties of the last century, local pig breeds in China are impacted by the introduction of pig breeds, and the breeding quantity is drastically reduced. In recent years, along with the improvement of the living standard of people, the demand for high-grade pork is increased, and local pig resources such as Min pigs are favored in the market again. However, due to the limitation of the current practical production level, the excellent germplasm characteristics of the Min pigs are not fully developed and utilized. The whole work of mining and identifying the excellent germplasm characteristics of the Min pig is relatively backward, and no mature related molecular identification method and standard exist, so that reasonable development and utilization of the Min pig are greatly hindered. The SNP chip is widely applied to livestock and poultry trait genetic analysis and breeding practice, but the SNP chip applied to pigs at present is designed aiming at introduced varieties and is not suitable for local pig breeds in China, so that the development of a genotyping detection product suitable for Min pigs is very in line with the development requirement of local pig industry in the present stage, and the protection of Min pig germplasm resources and the improvement of future breeding are effectively promoted.
Disclosure of Invention
The invention aims to provide a 15K liquid phase chip preparation method and a genotyping method based on the sequencing result of the whole genome of a Min pig for the Min pig population, and solves the problem that the current lack of local pig seed preservation and breeding chips and solid phase chips are inflexible.
The invention relates to a preparation method of a 15K liquid phase chip for Min pig breeding, which comprises a Min pig 15K probe mixed solution and a hybridization capture agent; the preparation method of the 15K liquid phase chip comprises the following steps:
step one, selecting Min pigs, extracting DNA of ear tissues and storing the DNA for later use;
step two, carrying out whole genome re-sequencing on the extracted Min pig ear tissue DNA, screening SNP loci at a whole genome level, and establishing a SNP locus set capable of covering Min pig groups to obtain 15000 SNP loci;
thirdly, respectively synthesizing two DNA nucleotide sequences with the length of 110bp and the 5' end with biotin gene modification according to the design principle of probes of a liquid phase capturing technology for the screened 15000 SNP loci, and defining the DNA nucleotide sequences as probes;
and step four, mixing the probes synthesized in the step three with equal molar mass, and utilizing EDTA and Tris-HCl mixed solution to fix the volume to obtain a 15K probe mixed solution, and combining the 15K probe mixed solution with a hybridization capture reagent to obtain the 15K liquid phase chip for Min pig breeding.
Further, the whole genome re-sequencing of the extracted Min pig ear tissue DNA in the second step means:
the method comprises the following steps of performing data processing on the re-sequencing original off-machine data of the extracted Min pig ear tissue DNA by using software fastp to obtain high-quality data, wherein the specific steps of the high-quality data acquisition are as follows: 1) Removing the linker sequence; 2) Removing paired read lengths with the N content exceeding 10% of the read length; 3) Removing paired read lengths with low mass base numbers exceeding 40% of the read length, wherein the low mass is Q less than or equal to 20; 4) A high quality clean read length is obtained.
Further, the step of obtaining the high-quality clean read length is to perform bioinformatics analysis on the read length from which the low-quality base number is removed, so as to obtain high-quality SNP data, and the specific steps are as follows: 1) Comparing the clean read length after quality control with a pig reference genome sequence by using software BWA, and positioning the position of the clean read length on the reference genome by comparison; the completion quality control means the reading length for removing the low-quality base number in the high-quality data acquisition; 2) According to the result of the step 1), a Haplotypeller module of the software GATK is used for detecting variation, and the detection parameters are as follows: GATK Best Practices; the filtration is performed using the variant filtration module of software GATK, the filtration parameters are: -filter-expression "QD <2.0||qual <30.0||mq <40.0||fs >60.0||sor > 3.0|mqrank sum < -12.5|readposrank sum < -8.0"; 3) And (3) carrying out position annotation on the basis of the SNP data set of the reference genome annotation file according to the annotation file of the reference genome (Sus Scrofa 11.1), and obtaining high-quality SNP locus data.
Further, in the second step, SNP loci are screened at the whole genome level, and the screening principle is as follows: the SNP loci are selected to be evenly distributed throughout the genome, and the minimum allele frequency of each SNP locus is greater than 0.35.
Further, in the third step, the probe design principle of the liquid phase capturing technology is followed, and the probe design principle is as follows: the length of the probe is 110bp; the GC content of the probe is between 30 and 70 percent, and the number of homologous regions is less than or equal to 5; designing two nucleotide sequences which have 60-70% overlap and cover the SNP locus of the SNP locus; the 5' end carries biotin gene modification.
Further, in the fourth step, the 15K probe mixture was fixed to a volume of 3pmol/mL by using a mixture of EDTA and Tris-HCl.
The genotyping method of the 15K liquid phase chip for Min pig breeding, which is prepared by the invention, is to enrich and capture SNP locus information of Min pig individuals to be detected, and genotype the individuals to be detected by utilizing the 15K liquid phase chip for Min pig breeding through high-throughput sequencing.
Further, the genotyping of the individuals to be tested by using the 15K liquid phase chip for Min pig breeding specifically comprises the following steps: firstly, extracting genomic DNA of a Min pig to be detected to construct a high-throughput sequencing library, then mixing a probe of a 15K liquid phase chip which can be used for Min pig breeding with the DNA high-throughput sequencing library of the Min pig to be detected, and capturing DNA fragments containing target sites in the DNA high-throughput sequencing library of the Min pig to be detected; amplifying and purifying the obtained DNA fragment, and carrying out high-throughput sequencing on the product, and analyzing by using BWA and GATK software to obtain SNP genotyping of the individual to be detected.
Further, the method for constructing the Min pig DNA high-throughput sequencing library to be tested comprises the following steps: fragmenting the Min pig genome DNA to be detected by adopting an enzyme digestion method, adding an A tail at the tail end, connecting a sequencing joint, and carrying out PCR amplification to obtain a Min pig DNA high-throughput sequencing library to be detected, and detecting the concentration of the Min pig DNA high-throughput sequencing library to be detected by utilizing the Qubit.
Further, the method for capturing DNA fragments containing target sites in the Minpig DNA high-throughput sequencing library to be tested comprises the following steps: and mixing a probe in a 15K liquid-phase chip containing biotin and capable of being used for breeding the Min pig with magnetic beads covered by streptavidin, adsorbing the Min pig genome DNA fragments to be detected on the magnetic beads through the combination of the biotin and the streptavidin, and washing out the DNA fragments in a non-target area through elution treatment so as to obtain the required Min pig genome DNA fragments to be detected.
Further, the hybridization capture reagent is a GenoBais DNA-seq Library Prep kit from Shimadzu Boruidi biotechnology Co., ltd, comprising GenoBaits Block I, genoBaits Block II, genoBais 2×HybBuffer, genoBaits Hyb Buffer Enhancer, genoBais 2× Beads Wash Buffer, genoBais 10×Wash Buffer I, genoBais 10×Wash Buffer II, genoBais 10X Wash Buffer III, genoBais 10X Stringent Wash Buffer packaged independently.
The invention has the following beneficial effects:
compared with the current SNP chip, the 15K liquid phase chip manufacturing method for the Min pig breeding is a specialized chip for the Min pig, so that the application of the method in the Min pig breeding can improve the seed selection accuracy and shorten the generation interval.
The Min pig 15K liquid phase chip is composed of a separately packaged Min pig 15K probe mixed solution and a hybridization capture reagent, wherein the Min pig 15K probe is a DNA double-stranded probe and is a nucleotide sequence designed and synthesized for SNP loci screened by Min pig whole genome sequences; the SNP locus is obtained by carrying out 12 x deep sequencing on the whole genome sequences of 30 pure-bred Min pigs, and comparing the whole genome sequencing results of the Min pigs to the SNP locus screening carried out on a pig reference genome (Sus Scrofa 11.1 version). The Min pig 15K liquid phase chip prepared by the method is mixed with a Min pig DNA high-throughput sequencing library to be detected, DNA fragments containing target sites in the Min pig DNA high-throughput sequencing library to be detected are captured for amplification and purification, and after high-throughput sequencing, sequencing results are returned to a pig reference genome for comparison, so that genome genotyping of the Min pig to be detected is obtained. The 15K liquid phase chip prepared by the invention can genotype individual Min pigs, can be used for the breeding and the breeding work of Min pigs, and solves the problem that the chip special for breeding local pig varieties is lacking in China at present.
The pig 50K liquid phase chip developed based on the preparation method and the application of the pig 50K liquid phase chip based on the targeted capturing sequencing is developed based on the introduced variety Du growing design, is not suitable for local pig varieties in China, is not only suitable for civilian pigs, but also can be popularized to other local pigs. Meanwhile, compared with 50K liquid phase chips, the number of SNP loci selected by the invention is reduced by 35000, the use cost is greatly reduced, and the genotyping requirement can be met.
Drawings
FIG. 1 is a distribution diagram of the number of SNP loci on different chromosomes in a Min pig 15K liquid chip provided by the invention.
Detailed Description
For the purposes of clarity, technical solutions and advantages of embodiments of the present invention, the spirit of the present disclosure will be described in detail below, and any person skilled in the art, after having appreciated the embodiments of the present disclosure, may make changes and modifications to the techniques taught by the present disclosure without departing from the spirit and scope of the present disclosure.
The exemplary embodiments of the present invention and the descriptions thereof are intended to illustrate the present invention, but not to limit the present invention.
Example 1
A15K liquid phase chip manufacturing method for Min pig breeding comprises the following steps:
step one, whole genome sequencing and data analysis of 30 pure-bred Min pigs;
the fungus tissue samples of 30 pure-bred Min pigs are collected at the Qing county Min pig breeding farm in Heilongjiang province and stored in a refrigerator at the temperature of minus 20 ℃ for standby.
The kit is used for extracting DNA of the ear tissue, and the required reagents comprise:
lysis buffer (10 mM Tris-HCl ph= 8.0;100mM EDTA pH =8.0) (laboratory formulation), DNA extraction kit (Qiagen), proteinase K (Shanghai geto Biochemical technologies limited), tris saturated phenol (biological engineering Co., ltd.), isoamyl alcohol (Beijing chloroform and absolute ethyl alcohol (Tianjin city, rich fine chemical Co.).
The specific steps are as follows:
1) Collecting 30 pure pig ear tissue samples, taking 10mg of each individual, respectively placing into 1.5mL EP tubes, and cutting with small scissors;
2) Adding 500 μl of tissue extract and 5 μl of proteinase K, shaking at 55deg.C and 120rpm overnight for digestion;
3) After digestion, 500. Mu.L of Tris saturated phenol is added and the mixture is turned upside down for 10min;
4) Centrifuging at 12000rpm for 10min, collecting upper water phase, and discarding lower water phase;
5) Adding an equal volume of phenol: chloroform: isoamyl alcohol (24:23:1) for 10min;
6) Centrifuging at 12000rpm for 10min, collecting upper water phase, and discarding lower water phase;
7) An equal volume of chloroform was added: isoamyl alcohol (23:1) for 10min; centrifuging at 12000rpm for 10min, collecting upper water phase, and discarding lower water phase;
8) 2 volumes of absolute glacial ethanol (-20 ℃) were added and shaken horizontally about 30 times to develop white flocs (DNA);
9) Centrifuging at 10000rpm for 10min;
10 700. Mu.L of 70% glacial ethanol is added, the mixture is centrifuged at 8000rpm for 5min, the ethanol is poured off and the mixture is naturally dried;
11 50. Mu.L of TE buffer was added and DNA was dissolved in a water bath at 55 ℃;
12 After the quality and the concentration are detected by a Nanodrop-100 spectrophotometer, the concentration is uniformly diluted to 50 ng/mu L and is preserved at the temperature of minus 20 ℃ for standby.
13 Quality and concentration detection is carried out on the extracted genome DNA, the concentration of the DNA is accurately quantified by using the Qubit, and DNA samples with the total amount of more than 200ng can be used for library establishment; agarose gel electrophoresis analysis of DNA degradation degree and whether RNA and protein contamination. All qualified samples are placed in a refrigerator at the temperature of-80 ℃ for standby.
And step two, delivering the DNA sample which is detected to be qualified in the step one to Shenzhen Dada company for sequencing, constructing a library for the synthetic sample, and then carrying out whole genome resequencing according to a company standard flow. Performing data processing on the re-sequenced original machine-down data by using fastp software to obtain high-quality data, wherein the data quality control principle is as follows:
1) Removing the linker sequence (adapter);
2) Removing paired read lengths with N content exceeding 10% of read length (reads);
3) Removing paired read lengths with the number of low-quality (Q is less than or equal to 20) bases exceeding 40% of the read length;
4) A high quality clean read length (clearready) is obtained.
Bioinformatics analysis is carried out on clearready to obtain high-quality SNP data, and the specific steps are as follows:
1) Comparing the clean reads after quality control with a reference genome (version 11.1) sequence by using software BWA (mem comparison method), and positioning the positions of the clean reads on the reference genome by comparison;
2) According to the comparison result of clear reads in a reference genome, a HaplotyCaller module of software GATK (version 4.0.4.0) is used for mutation detection, and detection parameters are as follows: GATK Best Practices. The filtration was performed using a variant filtration module with the following filtration parameters: -filter-expression "QD <2.0||qual <30.0||mq <40.0||fs >60.0||sor > 3.0|mqrank sum < -12.5|readposrank sum < -8.0";
3) Performing position annotation on the basis of the SNP data set according to the annotation file of the reference genome;
the average sequencing depth of 30 pure pig breeds is 11.99×, the number of reads compared with the reference genome accounts for 99.74% of the total clean reads, the average insert is 217bp, the proportion of base coverage depth in the reference genome is not less than 5×88.65%, and the proportion of base coverage depth in the reference genome is not less than 10× 56.95%. Finally, 24,944,573 high-quality SNP loci are obtained.
Step three, screening 15000 SNP loci, wherein the screening principle is as follows:
1) The length of the probe is 110bp;
2) The GC content of the probe is between 30 and 70 percent, and the number of homologous regions is less than or equal to 5;
3) The two sequences overlap 60-70% and cover the SNP locus; 18412 SNP loci are finally screened out.
Step four, selecting 186 pure pig individuals to extract DNA, and then constructing a library of the extracted qualified DNA samples and capturing the areas where the candidate 18412 SNP loci are located;
both the pooling and capture reagents were from the Genobaits DNA-seq library prep kit of Shimadzu borrelidi Biotechnology Co.
The concrete operation flow of library building is as follows:
1) The following reaction system was prepared in a PCR tube in a total amount of 20. Mu.L: 200ng of Min pig genome DNA, genoBaits End Repair Buffer mu L, genoBaits End Repair enzyme3.1 mu L and the balance of ultrapure water; the reagents used were from the GenoBaits DNA-seq Library Prep kit from Shijia Boruidi Biotechnology Co., ltd;
2) Slightly and uniformly mixing the reaction system, and collecting the reaction solution to the bottom of the tube by short centrifugation;
3) The reaction tube was placed in a PCR instrument and the following reactions were performed: a heat cover at 82 ℃, 20min at 37 ℃, 20min at 72 ℃ and 4 ℃ for preservation;
4) The following components were directly added to the above reaction tube: the total amount was 20. Mu.L, of which GenoBaits Ultra DNA Ligase. Mu.L, genoBaits ULtra DNA Ligase Buffer. Mu.L, genoBaits Adapter for MGI. Mu.L and the balance being ultrapure water;
5) After the reaction system is gently mixed, the reaction solution is collected to the bottom of the tube by short centrifugation;
6) The reaction tube was placed in a PCR instrument and the following reactions were performed, eliminating the hot cap: preserving at 22deg.C for 60min and 4deg.C;
7) Adding 48 mu L of DNA purified magnetic beads which are balanced for more than 30 minutes at room temperature into the system, vibrating and uniformly mixing, keeping the mixture stand for 5 minutes without generating bubbles as much as possible, and collecting liquid to the bottom of a tube by short centrifugation;
8) Placing on a magnetic rack for at least 3min until the solution is clear, and removing the supernatant;
9) Keeping the PCR tube in a magnetic rack, adding 100 mu L of 80% ethanol, incubating for 30s at room temperature, and removing the supernatant;
10 Maintaining the PCR tube in the magnetic frame, uncovering and airing for 5min until the ethanol volatilizes;
11 Taking the PCR tube out of the magnetic frame and airing the magnetic beads in the PCR tube;
12 Preparing the following reaction system in the new PCR tube: the total amount was 20. Mu.L, of which GenoBa its PCR Master Mix 10.mu. L, I5.5 Barcode (10 μm) -MGI 1.mu. L, I7 Barcode (2 μm) -MGI 5. Mu.L, and ultrapure water 4. Mu.L; adding the system into the PCR tube of 11), re-suspending the dried magnetic beads, and briefly centrifuging to collect the reaction solution to the bottom of the tube;
13 The reaction tube was placed in a PCR instrument and the following reaction was performed: 2min at 98 ℃,6-8 cycles: 98 ℃ for 30s,50 ℃ for 30s,72 ℃ for 40s, and finally 72 ℃ for 4min;
14 Adding 20 mu L of DNA purified magnetic beads which are balanced for more than 30 minutes at room temperature into the system, vibrating and uniformly mixing, avoiding generating bubbles as much as possible, standing for 5 minutes, and collecting liquid to the bottom of a tube by short centrifugation;
15 Placing the PCR tube on a magnetic rack for at least 3min until the solution is clear, and removing the supernatant;
16 Maintaining the PCR tube in a magnetic rack, adding 100 mu L of 80% ethanol, incubating at room temperature for 30s, and removing the supernatant;
17 Keeping the PCR tube in the magnetic rack, uncovering and airing for 10min.
18 Taking the PCR tube out of the magnetic frame, adding 35 mu L of Tris-HcL, shaking and uniformly mixing, standing for 5min, and collecting the liquid to the bottom of the tube by short centrifugation;
19 Placing the PCR tube on a magnetic rack, clarifying the solution (about 3 min), and transferring the supernatant to a new tube to complete the construction of the DNA sequencing library.
20 Library quality detection: taking 1 mu L of the prepared library sample, quantifying the library sample by using Qubit, and sequencing the total DNA amount of the library to be more than 500 ng; 3. Mu.L of the prepared library sample was taken and detected by 1% agarose gel electrophoresis, and the result showed that the DNA fragment was 300-500bp. The prepared library samples were stored at 4 ℃.
The specific capturing operation flow is as follows:
1) Balancing the DNA purification magnetic beads for more than 30min at room temperature, uniformly mixing the Min pig 15K probe and the DNA purification magnetic beads in a 0.2mL PCR tube, and centrifuging for a short time;
2) The following reagents were mixed in the PCR tube: 500 ng/library of the Min pig DNA high-throughput sequencing library to be tested, genoBaits BLock I mug (5 mug), genoBaits Lock II for ILM/MGI 2 mug and 300ng of the probe to be tested, so as to form a liquid phase chip mixed solution containing the Min pig DNA high-throughput library to be tested and the Min pig 15K probe;
3) Concentrating the PCR tube to be completely dry by using a vacuum concentrator at the temperature of less than or equal to 60 ℃;
4) Centrifuging the PCR tube 12000rpm for 1min after the concentration is finished;
5) All GenoBaits hybridization reagents were dissolved at room temperature; if GenoBaits 2 XHyb Buffer is crystallized, heating to 65deg.C, and shaking for a while until completely dissolving;
6) The following reagent systems were added to the finished PCR tubes: 8.0. Mu.L GenoBaits 2 XHyb Buffer, 2.0. Mu. L GenoBaits Hyb Buffer Enhancer, 6.0. Mu.L NucLease-Free Water;
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 an EP tube of 0.2 mL;
8) Incubating the EP tube in a PCR amplification apparatus at 95℃for 10min (heat cover temperature 105 ℃);
9) And transferring the EP tube to another 65-DEG C PCR instrument with a 75-DEG C heat cover when the temperature of the PCR amplification instrument is reduced to 65 ℃, and carrying out a warm bath for 1h.
10 The eluent needing to be preheated at 65 ℃ is put into a PCR instrument in advance, and the rest eluent is stored at room temperature. The eluent needs to be prepared in advance, as follows: (1) 250. Mu.L of GenoBaits 2X Beads Wash Buffer plus 250. Mu.L of water; (2) 28. Mu.L of GenoBaits10 XWash Buffer I plus 252. Mu.L of water; (3) 18. Mu.L of GenoBaits10 XWash Buffer II with 162. Mu.L of water; (4) 18. Mu.L of GenoBaits10 XWash Buffer III with 162. Mu.L of water; (5) 40. Mu.L of GenoBaits 10X Stringent Wash Buffer plus 360. Mu.L of water; (6) 250. Mu.L of GenoBaits 2X Beads Wash Buffer. Mu.L with 500. Mu.L of water;
11 Preparing DNA purification magnetic beads, wherein the DNA purification magnetic beads are ready to use and are prepared by the following steps: the DNA purification magnetic beads are placed for 10min at room temperature before being used, and are uniformly mixed by vortex oscillation for 15 s;
12 Prepare 50. Mu.L of DNA purification beads in 0.2mL of EP tube, place EP tube on a magnetic rack, and allow the beads to separate thoroughly from the solution. Removing the supernatant and retaining the magnetic beads;
13 In the above EP tube, 150. Mu.L of GenoBaits1 XBead Wash Buffer was added, vortexed for 10s, the EP tube was transferred to a magnetic rack, the beads were completely separated from the solution, the supernatant was removed, and the washing was repeated 2 times for 3 times altogether;
14 Transferring 16. Mu.L of hybridization solution in a temperature bath at 65 ℃ for 1h into the prepared EP tube, operating on a PCR instrument, shaking by vortex for 10s, fully mixing, and centrifuging for 5s.
15 Placing the EP tube into a PCR instrument, heating to 75deg.C for 45min, combining DNA and magnetic beads every 12min, and vibrating for 5s.
16 100. Mu.L of preheated GenoBaits1 XWash Buffer I at 65℃was added to each EP tube, vortexed briefly for 5s, centrifuged for 5s, and then placed on a magnetic rack with the beads completely separated from the solution, removing the supernatant (unbound DNA);
17 150. Mu.L of preheated (65 ℃) GenoBaits 1X Stringent Wash Buffer was slowly pipetted up and down 10 times (avoiding air bubbles); after the last group of samples are uniformly mixed, the samples are placed for 2 minutes (the time requirement is accurate); placing on a magnetic rack, completely separating magnetic beads from the solution, rapidly removing supernatant by using a pipette, and repeating the operation for 1 time;
18 150. Mu.L of GenoBaits1 XWash Buffer I at room temperature, shaking for 2min, placing on a magnetic frame, completely separating magnetic beads from the solution, and removing the supernatant;
19 150. Mu.L of GenoBaits1 XWash Buffer II at room temperature is added, the mixture is vibrated for 1min and placed on a magnetic frame, the magnetic beads are completely separated from the solution, and the supernatant is removed;
20 150. Mu.L of GenoBaits1 XWash Buffer III at room temperature is added, the mixture is oscillated for 30s, the magnetic beads are completely separated from the solution on a magnetic rack, and the supernatant is removed;
21 Removing the tube containing the captured DNA from the magnetic rack; adding 20 mu L of NucLease-Free Water, sucking for 10 times, ensuring that all magnetic beads are resuspended, taking care not to discard the magnetic beads, using 10 mu L of the magnetic bead resuspension for the following PCR amplification and purification, and leaving the rest 10 mu L as a backup;
amplifying and purifying the obtained DNA fragment, and after high-throughput sequencing, replying the product to a pig reference genome by using a sequencing result for comparison, thereby obtaining the genome genotyping of the pig to be tested.
1) Depending on library type, PCR reagents were prepared in 0.2mL PCR tubes: genoBaits PCR Master Mix 15. Mu.L, genoBaits Primer Mix for MGI 1.2.2. Mu.L, 10. Mu.L of captured DNA beads, 3.8. Mu.L of ultrapure water, and a total of 30. Mu.L;
2) Short vortex and centrifugation ensure that the magnetic beads are still in solution;
3) The PCR tube was placed in a PCR apparatus at a hot lid temperature of 105℃and PCR amplification was performed according to the following procedure: pre-denaturation at 98℃for 45s; amplification for 12 cycles: 15s at 98 ℃, 30s at 50 ℃ and 30s at 72 ℃; extending at 72℃for 1min. Preserving at 4 ℃;
4) Adding 45 mu L (1.5 times of volume) of magnetic beads with captured DNA into each PCR reaction, shaking and mixing uniformly, keeping the mixture stand for 5min without generating bubbles as much as possible, and collecting the liquid to the bottom of a tube by short centrifugation; placing on a magnetic rack for at least 3min until the solution is clear, and removing the supernatant;
5) 45 μl (1.5 times volume) of GenoPrep DNA Clean Beads was added to each PCR reaction, mixed by shaking to avoid air bubbles as much as possible, left to stand for 5min, and the liquid was collected to the bottom of the tube by brief centrifugation.
6) Placing on a magnetic rack for at least 3min until the solution is clear, and removing the supernatant;
7) Keeping the PCR tube in a magnetic frame, adding 100 mu L of 80% ethanol, incubating at room temperature for 30s, removing the supernatant, keeping the PCR tube in the magnetic frame, and uncovering and airing for 10min;
8) Taking out the PCR tube from the magnetic rack, adding 35 mu L of Tris-HcL, shaking and uniformly mixing, standing for 5min, and collecting the liquid to the bottom of the tube by short centrifugation; transferring supernatant to new tube after solution is clarified (about 3 min) on magnetic rack, preserving at-20deg.C, and preserving purified PCR fragment at-20deg.C for one week;
9) The library was measured using Qubits FLuorometer and Qubit dsDNA HS Assay Kit and the average length of the captured DNA library fragments on the digital electrophoresis system was measured; library concentrations were measured with KAPA Library Quantification Kit.
10 Probe sequencing with a sequencer.
As can be seen from FIG. 1, the SNP loci selected in the present invention are uniformly distributed on 18 chromosomes of a pig.
The detection results of the test sample are shown in table 1, and the detection rate is as follows: the average detection rate of SNP of 186 samples is 99.57%, and the standard deviation is 0.041. The maximum value and the minimum value of the individual detection rate are 99.22 percent and 92.64 percent respectively, and the average detection rate is 98.74 percent; the liquid phase chip has good genotype detection quality.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. The preparation method of the 15K liquid phase chip for Min pig breeding is characterized in that the liquid phase chip consists of a Min pig 15K probe mixed solution and a hybridization capture agent; the preparation method of the 15K liquid phase chip comprises the following steps:
step one, selecting Min pigs, extracting DNA of ear tissues and storing the DNA for later use;
step two, carrying out whole genome re-sequencing on the extracted Min pig ear tissue DNA, screening SNP loci at a whole genome level, and establishing a SNP locus set capable of covering Min pig groups to obtain 15000 SNP loci;
thirdly, respectively synthesizing two DNA nucleotide sequences with the length of 110bp and the 5' end with biotin gene modification according to the design principle of probes of a liquid phase capturing technology for the screened 15000 SNP loci, and defining the DNA nucleotide sequences as probes;
and step four, mixing the probes synthesized in the step three with equal molar mass, and utilizing EDTA and Tris-HCl mixed solution to fix the volume to obtain a 15K probe mixed solution, and combining the 15K probe mixed solution with a hybridization capture reagent to obtain the 15K liquid phase chip for Min pig breeding.
2. The method for preparing a 15K liquid-phase chip for Min pig breeding according to claim 1, wherein the step two of performing whole genome re-sequencing on the extracted Min pig ear tissue DNA means:
the method comprises the following steps of performing data processing on the re-sequencing original off-machine data of the extracted Min pig ear tissue DNA by using software fastp to obtain high-quality data, wherein the specific steps of the high-quality data acquisition are as follows: 1) Removing the linker sequence; 2) Removing paired read lengths with the N content exceeding 10% of the read length; 3) Removing paired read lengths with low mass base numbers exceeding 40% of the read length, wherein the low mass is Q less than or equal to 20; 4) A high quality clean read length is obtained.
3. The preparation method of the 15K liquid phase chip for Min pig breeding according to claim 2, wherein the method is characterized in that the method is used for obtaining high-quality clean read length, and the read length from which low-quality base numbers are removed is subjected to bioinformatics analysis to obtain high-quality SNP data, and comprises the following specific steps: 1) Comparing the clean read length after quality control with a pig reference genome sequence by using software BWA, and positioning the position of the clean read length on the reference genome by comparison; the completion quality control means the reading length for removing the low-quality base number in the high-quality data acquisition; 2) According to the result of the step 1), a Haplotypeller module of the software GATK is used for detecting variation, and the detection parameters are as follows: GATK Best Practices; the filtration is performed using the variant filtration module of software GATK, the filtration parameters are: -filter-expression "QD <2.0||qual <30.0||mq <40.0||fs >60.0||sor > 3.0|mqrank sum < -12.5|readposrank sum < -8.0"; 3) And carrying out position annotation on the basis of the SNP data set of the annotation file of the reference genome according to the annotation file of the reference genome, and obtaining high-quality SNP locus data.
4. The preparation method of a 15K liquid phase chip for Min pig breeding according to claim 1, wherein in the second step, SNP loci are screened at the whole genome level, and the screening principle is as follows: the SNP loci are selected to be evenly distributed throughout the genome, and the minimum allele frequency of each SNP locus is greater than 0.35.
5. The preparation method of the 15K liquid phase chip for Min pig breeding according to claim 1, wherein in the third step, the probe design principle of the liquid phase capturing technology is followed, and the probe design principle is as follows: the length of the probe is 110bp; the GC content of the probe is between 30 and 70 percent, and the number of homologous regions is less than or equal to 5; designing two nucleotide sequences which have 60-70% overlap and cover the SNP locus of the SNP locus; the 5' end carries biotin gene modification.
6. The method for preparing a 15K liquid phase chip for Min pig breeding according to claim 1, wherein in the fourth step, the 15K probe mixed solution with the constant volume of 3pmol/mL is prepared by using EDTA and Tris-HCl mixed solution.
7. The genotyping method of the 15K liquid phase chip for Min pig breeding according to any one of claims 1 to 6, wherein the genotyping method is characterized in that SNP site information of Min pig individuals to be detected is collected in an enriched mode, and the individuals to be detected are subjected to genotyping by utilizing the 15K liquid phase chip for Min pig breeding through high-throughput sequencing.
8. The genotyping method according to claim 7, wherein the genotyping of the individual to be tested by using the 15K liquid phase chip for Min pig breeding specifically comprises: firstly, extracting genomic DNA of a Min pig to be detected to construct a high-throughput sequencing library, then mixing a probe of a 15K liquid phase chip which can be used for Min pig breeding with the DNA high-throughput sequencing library of the Min pig to be detected, and capturing DNA fragments containing target sites in the DNA high-throughput sequencing library of the Min pig to be detected; amplifying and purifying the obtained DNA fragment, and carrying out high-throughput sequencing on the product, and analyzing by using BWA and GATK software to obtain SNP genotyping of the individual to be detected.
9. The genotyping method according to claim 8, wherein the method for constructing the Min pig DNA high throughput sequencing library to be tested comprises the following steps: fragmenting the Min pig genome DNA to be detected by adopting an enzyme digestion method, adding an A tail at the tail end, connecting a sequencing joint, and carrying out PCR amplification to obtain a Min pig DNA high-throughput sequencing library to be detected, and detecting the concentration of the Min pig DNA high-throughput sequencing library to be detected by utilizing the Qubit.
10. The genotyping method according to claim 8, wherein the method for capturing DNA fragments containing target sites in the Min pig DNA high throughput sequencing library comprises the steps of: and mixing a probe in a 15K liquid-phase chip containing biotin and capable of being used for breeding the Min pig with magnetic beads covered by streptavidin, adsorbing the Min pig genome DNA fragments to be detected on the magnetic beads through the combination of the biotin and the streptavidin, and washing out the DNA fragments in a non-target area through elution treatment so as to obtain the required Min pig genome DNA fragments to be detected.
CN202311488548.0A 2023-11-09 2023-11-09 Preparation method of 15K liquid phase chip for Min pig breeding and genotyping method Pending CN117363750A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117701722A (en) * 2024-01-16 2024-03-15 佛山科学技术学院 Cattle plateau adaptive breeding 10K liquid phase chip and application

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117701722A (en) * 2024-01-16 2024-03-15 佛山科学技术学院 Cattle plateau adaptive breeding 10K liquid phase chip and application

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