CN117778588A - Preparation method and application of sheep 1K liquid phase chip based on targeted capturing sequencing - Google Patents
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Abstract
The invention relates to a preparation method and application of a sheep 1K liquid phase chip based on targeted capture sequencing, wherein the sheep 1K liquid phase chip is composed of an independently packaged sheep 1K probe mixed solution and a hybridization capture reagent, the sheep 1K probe is a DNA double-stranded probe, and site information of the sheep 1K probe relates to 1088 SNP sites. The sheep 1K liquid phase chip prepared by the method is mixed with a sheep DNA high-throughput sequencing library, and the products are captured, amplified and purified, and compared after high-throughput sequencing, so that the genome genotyping of the sheep to be detected is obtained. The sheep 1K liquid phase chip site provided by the invention comprises 1088 SNP markers and 1Y chromosome DNA fragments to prepare a 1K liquid phase chip for genotyping the lambs. Has an open meaning in the aspect of sheep breeding and breeding, can solve the problems that the prior art has high cost, is inflexible and can not be used in a large scale in sheep farms, and greatly improves the sheep breeding and breeding process.
Description
Technical Field
The invention relates to the technical field of genotyping, in particular to a preparation method of a sheep 1K liquid chip based on a targeted capture sequencing genotyping (Genotyping by Targeted Sequencing, GBTS) technology and application of the sheep 1K liquid chip in genotyping of sheep genome.
Background
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to a polymorphism in a DNA sequence caused by a single nucleotide variation on the genome, and the presence of transitions, transversions, deletions and insertions. SNP is the most common genetic polymorphism, the number is large, the distribution is wide and dense, the proportion of the SNP accounts for more than 90% of all known polymorphisms, and the SNP is used as a third generation genetic marker and has the characteristics of easy detection, easy rapid large-scale screening, ideal genotyping, two-state property and the like, and is one of main research contents of the human genome era. With the development of high-throughput sequencing technology, a large number of SNP markers are discovered and widely applied to various fields of biology, agriculture, medicine and the like. A large number of SNP markers can be used for research of animal and plant gene functions and association analysis of diseases, and the phenotype difference is explained by comparing SNP differences among individuals.
Along with the lower price and wider application range of SNP chips, the SNP chip technology has entered a new era, and the development of the technology makes the screening speed of the SNP chip faster, the coverage wider and the economic cost lower, and the SNP chip technology is widely applied to genetic analysis such as animal and plant population genetic analysis, genetic relationship and variety identification. In recent years, various SNP chips are rapidly developed, widely applied to animals and plants, and have great significance in researching germplasm characteristics, genetic breeding, diseases and the like. SNP chips include solid phase chips and liquid phase chips. A solid-phase SNP chip, namely a single nucleotide polymorphism microarray (SNP Array), is a SNP probe Array formed by arraying millions of DNA marker sequences on a glass slide or a special silicon wafer and fixing. The working principle is that the DNA marker sequence fixed on the chip and the target genome are subjected to base complementary pairing reaction, so that the genetic variation information is accurately identified. SNP chips play a central role in the release of third generation human genome maps and mutation data. At present, two solid-phase SNP chips are mainly popular in the market, and have been widely applied to the detection of genome variation of animals and plants. One is a high-density SNP chip based on oligonucleotide in-situ lithography patent technology by ThermoFisher company (original Affymetrix company), which is widely applied to association analysis (association), copy number variation analysis (copy number variations, CNV), linkage analysis (linkage), gene expression and spliceosome analysis, epigenetic analysis and the like; another is the Illumina Infinium chip from Illumina, inc. based on fiber-optic microbeads, which are formed by coupling specific gene probe sequences to microbeads 3 μm in diameter, self-assembling in the microwells of the matrix. However, the existing solid-phase chip can only type SNP loci contained on the chip, SNP loci surrounding the loci cannot be typed, loci which can be detected once designed are fixed, the loci cannot be added and deleted, and the flexibility is poor. Liquid phase chip is a chip technology based on the principle of liquid phase hybridization. It detects SNP sites in a sample by dissolving a series of specific DNA probes in a liquid phase and then hybridizing DNA fragments in the sample to be detected with these probes. The liquid phase SNP chip has the advantages of low cost, high flux, high sensitivity, high specificity and the like, has wider application range, and can be used for molecular marker assisted selection, whole genome selection and other application scenes. At present, a boriding sheep 40K liquid phase chip, a 'middle core breeding sheep No. 1' 5K liquid phase chip, a 'yellow triangle snowflake first' sheep gene chip and a 'XingYi' 20K sheep breeding chip have been widely used for large-scale commercial breeding of sheep, and specific applications comprise genetic diversity analysis, variety relation analysis, whole genome association analysis, quantitative trait positioning analysis of QTL, selective evolution research and genome selection.
In addition, while the use of high density SNP chips can improve the accuracy of breeding value estimation to some extent, the high genotyping costs greatly increase the breeding costs, limiting the widespread use of whole genome selection in sheep breeding, and the desire for higher density markers is also increasing the cost of genotyping. In sheep breeding, high-density SNP chip genotyping and genome breeding value prediction are carried out on candidate stud rams with higher market value, and the high genotyping cost is acceptable, but the genotyping or resequencing of the high-density SNP chip is carried out on the whole beef cattle population to obtain genotype data, so that the mutton sheep breeding cost is increased sharply, and the economic benefit of breeding enterprises is reduced. On the other hand, prediction of genomic breeding values using high density markers would increase computational time and difficulty and place high demands on the hardware and software of the computing device. For example, in the calculation of genomic estimated breeding values, the solution of equations in predictive models is difficult because the number of phenotypes is significantly smaller than the number of markers and the group expansion rate of the reference population is much lower than the rate of increase of the markers. In general, the use of high-density markers for whole genome selection not only brings pressure to the calculation of genome estimated breeding values and challenges the development of new algorithms, but also greatly increases the investment of breeding cost and influences the economic benefits of breeding, thus having certain limitations.
In contrast to the current sheep 1K chip, namely a liquid chip, which is mainly used in the market, and the solid-phase chip developed based on an illumine platform and an Affymetrix platform, the method has the advantages of being rich in property association sites, completely independent in domestic intellectual property rights and free of political trade risks, besides the advantages of high detection accuracy and lower price; the density of the developed SNP chip can be flexibly adjusted according to the improvement process, new sites can be replaced or 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 and medium-high density liquid-phase chip.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method and application of a sheep 1K liquid phase chip based on targeted capturing sequencing, and simultaneously provides a special SNP probe design principle and a sheep whole genome 1K liquid phase chip.
The invention provides a preparation method of a sheep 1K liquid phase chip based on targeted capture sequencing, which comprises the following steps:
(1) Screening 1088 SNP loci designed and synthesized nucleotide sequences as probes; the 5' end of the probe is modified by biotin groups, and the modified probe is called a sheep 1K probe;
(2) Mixing the synthesized sheep 1K probe with equal molar mass, and forming sheep 1K probe mixed solution by using mixed solution of EDTA and Tris-HC 1;
(3) And packaging the sheep 1K probe mixed solution and the hybridization capture reagent respectively and independently to obtain the sheep 1K liquid phase chip based on targeted capture sequencing.
The preparation method as described above, preferably, the principle of screening SNP loci comprises the following steps: step S1, determining the phenotype value of the main economic character of sheep, and identifying the genotype of the genomic genetic variation of the corresponding individual by utilizing a genomic resequencing technology;
s2, arranging phenotype, pedigree and fixed effect files;
s3, estimating a breeding value by using the phenotype, the pedigree and the fixed effect, and calculating a corrected phenotype; s4, quality control is carried out on the re-sequencing data, wherein the quality control standard is that SNP loci with SNP detection rate less than 30%, minimum allele frequency less than 0.05, minimum average sequencing depth less than 5 and no chromosome position information are removed; step S5, selecting the corrected phenotype and the resequenced data of the resequenced individual for genome wide association analysis (GWAS); performing whole genome association analysis on the main character indexes by using a Mixed Linear Model (MLM), setting a statistical test threshold value of genome level significance by using a Bonferroni method, and judging significant SNPs associated with each character; finally, selecting SNP loci with the top 0.00001% of each character significance rank as candidate SNP loci which are obviously related to the characters;
S6, selecting the corrected phenotype and the resequenced data of the resequenced individuals from the resequenced data obtained in the step S4, estimating the effect value of the reference number group on the SNP marker by using a rrBLUP method, and respectively detecting and explaining the SNP loci with larger proportion of the additive genetic variance of the main economic characters; calculating to obtain the effect value of the SNP marker of the corresponding character, and selecting SNP loci with the interpretation additive genetic variance proportion ranking of 0.00001% as candidate loci;
step S7, sliding window screening is carried out on the re-sequenced data after quality control obtained in the step S4; sliding a window with 25000Kb length on an autosome by using the oar_rambouille_v1.0 sheep genome assembly result, and screening information SNP loci with the highest MAF in the window, wherein the SNP loci can be uniformly distributed at intervals on the genome; specific screening criteria are as follows: 1) Selecting the SNP locus with the highest MAF in a 25000Kb sliding window; 2) SNP loci with 80% detection rate to ensure stable reproducibility of loci; finally, in order to avoid the lack of flanking marker information at the beginning/end of each chromosome, the first and last SNPs of each chromosome must be selected; thus, for each constant stain, the highest MAF SNP site in each 25000Kb window was selected and the SNP at the beginning and end of each chromosome was included;
S8, carrying out re-sequencing on the quality-controlled data obtained in the step S4, downloading an oar_rambouille_v1.0 sheep genome annotation file from an Ensemble database, and annotating high-quality SNP loci to corresponding gene areas according to the physical positions of the markers, wherein the genes comprise protein coding genes, non-protein coding genes and partial pseudogenes; meanwhile, identifying a Hu sheep genome promoter, an enhancer, a chromatin opening region regulatory element and an active region based on ChIP-seq, ATAC-seq and CUT & Tag technologies; for each gene region, genome promoter, enhancer, chromatin opening region and other regulatory elements and active region, the SNPs annotated to that region will form a single set of SNPs, and the SNP site with the highest MAF is selected from the set;
step S9, screening specific loci among varieties: respectively calculating group fixed indexes (Fst) between Hu sheep and sheep of other varieties in a whole genome range by utilizing the collected multi-platform genotyping data of sheep variety resource materials, and selecting a window of the first 1 permillage of the highest Fst in the whole genome range to take intersection as a significantly selected region;
step S10, important SNP loci which are reported by an OMIA database (https:// www.omia.org/home /) and an Ishaep QTLdb database and related to sheep economic traits;
Step S11, combining SNP loci screened by the four methods (removing repeated screening loci) together to form a sheep 1K liquid phase chip SNP set;
step S12, calculating the interval, linkage disequilibrium and minimum allele frequency between adjacent SNP loci of the functional SNP locus chips in the step S11, and generating a final sheep 1K liquid phase chip locus set after eliminating abnormal loci, wherein the SNP loci are shown in serial numbers 1-1088;
step S13, selecting 1 DNA region design probe located on sheep Y chromosome for sex identification: the nucleotide sequence of the DNA region is shown as SEQ ID NO. 1;
the coverage area of the screened SNP locus simultaneously meets the design principle of sheep 1K probes.
The preparation method is preferably characterized in that the design principle of the sheep 1K probe is as follows: the length of the probe is 110bp, the GC content of the probe is between 30% and 70%, the number of homologous regions is less than or equal to 5, and the selected region does not contain an SSRX region and a GAP region to the maximum extent; two complementary sequences designed according to the upstream and downstream sequences of each SNP locus obtained by screening, wherein the two complementary sequences have nucleotide sequences which overlap 60-70% and cover the SNP locus; synthesizing single-stranded nucleotide from the designed nucleic acid sequence, wherein the synthesized two nucleic acid sequences are 110bp in length; the DNA nucleotide sequence modified by biotin group at the 5' end is called sheep 1K probe.
Preferably, the liquid-phase chip further comprises a probe shown as SEQ ID NO.2-SEQ ID NO.9, and the 5' end of the probe is modified by a biotin group.
Preferably, the hybridization capture reagent comprises an independently packaged GenoBaits DNAseq Library Prep kit, and specifically comprises independently packaged 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, and GenoBaits 10× Stringent Wash Buffer.
Preferably the sheep reference genome is oar_rambouillet version 1.0.
The invention provides a sheep genome genotyping method based on targeted capture sequencing, which comprises the following steps:
step 1, a sheep 1K liquid phase chip based on targeted capturing sequencing, which is prepared by adopting the preparation method, is adopted;
step 2, constructing a sheep DNA high-throughput sequencing library by utilizing genome DNA of sheep to be detected;
step 3, mixing the sheep 1K liquid phase chip prepared in the step 1 with the sheep DNA high-throughput sequencing library constructed in the step 2, and capturing DNA fragments containing target sites in the sheep DNA high-throughput sequencing library;
And 4, amplifying and purifying the DNA fragment obtained in the step 3, and after high-throughput sequencing, replying the product to a sheep reference genome by using a sequencing result for comparison, thereby obtaining the genome genotyping of the sheep to be tested.
The method as described above, preferably, in step 2, the method of constructing a sheep DNA high throughput sequencing library is: the method comprises the steps of fragmenting genome DNA of sheep to be detected by adopting an ultrasonic breaking method, filling and repairing the tail end of a fragment, adding an A tail, connecting a sequencing joint, purifying a connection product, carrying out fragment screening by using magnetic beads, retaining the connection product of an insert fragment of 200-300 bp, and carrying out PCR amplification by using the sequencing joint with a Barcode sequence and a P5 joint to obtain a sheep DNA high-throughput sequencing library.
Further preferably, in step 3, the capturing method is: the sheep 1K probe and the magnetic beads covered with streptavidin are mixed, the target sheep genomic DNA fragment is adsorbed onto the magnetic beads through the combination of the biotin and the streptavidin, and then the DNA fragment of a non-target region is washed out through elution treatment, so that the required sheep genomic DNA fragment is obtained.
Further preferably, in step 4, high throughput sequencing data is processed through SAMTOOLS, BWA, GATK and basenumber ngs software platforms to obtain SNP genotyping for a specific site in an individual.
The invention provides an application of sheep 1K liquid phase chip in sheep molecular breeding.
The sheep 1K liquid phase chip provided by the invention consists of an independently packaged sheep 1K probe mixed solution and a hybridization capture reagent, wherein the sheep 1K probe is a DNA double-stranded probe and is a nucleotide sequence designed and synthesized according to SNP loci; the sheep 1K probe site information is shown as the numbers 1-1088, and is obtained by comparing the whole genome sequencing result of sheep to the sheep reference genome to screen SNP sites. The sheep 1K liquid phase chip prepared by the method is mixed with a sheep DNA high-throughput sequencing library, DNA fragments containing target sites in the sheep DNA high-throughput sequencing library are captured for amplification and purification, and after high-throughput sequencing, the products are mapped onto a sheep reference genome by using a sequencing result for comparison, so that genome genotyping of sheep to be detected is obtained.
Compared with the prior art, the sheep 1K liquid phase chip based on targeted capturing sequencing and the sheep genome genotyping method provided by the invention have the following beneficial effects:
1. The invention provides a sheep high-throughput SNP 1K probe for genotyping based on targeted capture sequencing, wherein SNP loci which are obviously associated with sheep important traits are considered in the process of probe design; the global first group of large-scale sheep (n=2074) with multi-scale and multi-dimensional phenotype group records constructed by the sites depends on research and development teams, mainly determines main economic characters such as feed efficiency, growth, organism composition, meat quality, reproduction, immunity and the like, and can significantly improve the accuracy of genome breeding value estimation by mining and identifying 72.5 ten thousand pieces of high-quality phenotype data and 50T resequencing data of 250 important economic characters such as obtained feed efficiency, growth, organism composition, meat quality, reproduction, immunity and the like through whole genome association analysis (GWAS).
2. The invention provides a sheep high-throughput SNP 1K probe for genotyping based on targeted capture sequencing, wherein the distribution of captured SNP loci (global representativeness of background loci) in the whole genome is considered in the probe design, and the problem of locus polymorphism is considered in the capture probe, so that MAF requirement is greater than 0.40 in the Hu sheep population of the sheep variety of the current family in China, and the problems of uneven marker density and poor polymorphism possibly caused by simplifying the genome sequencing typing technology are effectively avoided.
3. According to the invention, 110bp before and after each SNP marker locus is taken, 2 probes are designed, the GC content of the probes is controlled between 30% and 70%, the number of homologous regions is less than or equal to 5, the hybridization stability of the sequence of each SNP is effectively improved, and the fragment capturing efficiency is improved. Meanwhile, the specific specificity of the flanking sequences of the sites on the genome is fully considered, the site specific capture rate is ensured, and the sequencing cost is reduced. The invention can design a capture probe for any specific SNP locus theoretically, so the invention has important application in large-scale SNP typing of sheep specific markers, and provides high-efficiency genome typing technology for genetic diversity analysis, strain identification, sex determination, marker-assisted breeding research and application of sheep.
4. The sheep 1K liquid phase chip can supplement SNP loci at any time, and new probes are designed according to the new loci and added into the existing sheep 1K probe mixed liquid. The invention can remove unnecessary SNP markers, does not carry out sequencing detection, adopts solid phase chip marker loci to fix in the prior art, can realize SNP locus increase and decrease only by designing new chips, and generally needs to consider new chip designs after the existing chips are used up for economic benefit. In addition, the solid-phase chip technology has requirements on sample size, and one chip can usually make 12, 24 or 48 or even 96 samples, so that the requirements on sample size are met, but the invention has no such requirements, the whole experiment is carried out in a PCR tube for reaction, a reaction system can be added according to the requirements, so that 1 sample can be detected, the sample detection size is flexible, the technical defects of the solid-phase chip are overcome, and the method has more popularization value in sheep molecular breeding.
5. According to the invention, by optimizing the hybridization capturing system, the S3-step DNA hybridization capturing time is 1 hour, compared with the overnight hybridization capturing process over 16 hours, the genotype capturing time can be greatly shortened, the whole process of database construction and capturing can be completed in a day, and the time for genotype detection is saved.
6. The probe has better tolerance to flanking sequences, so that the sheep 1K SNP liquid phase chip has higher tolerance to the variation condition of the flanking sequences, and can stably capture the target sequence under the condition that the flanking sequence variation is not higher than 10%, so that 80bp sequence information including up to 200K SNP variation information can be obtained at the upstream and downstream in addition to the target SNP information, and more information support is provided for sheep molecular breeding under the condition of the same cost.
Drawings
FIG. 1 shows the distribution of the number of SNP markers on different chromosomes in sheep 1K liquid phase chip provided by the invention.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
Example 1 sheep 1K liquid phase chip based on targeted capture sequencing and method applied to sheep genome genotyping
Step 1, preparing a sheep 1K liquid phase chip based on targeted capturing sequencing, wherein the sheep 1K liquid phase chip consists of a sheep 1K probe mixed solution and a hybridization capturing reagent which are independently packaged, and the preparation of the sheep 1K probe is a DNA double-stranded probe, and is a nucleotide sequence designed and synthesized according to a screened SNP locus; the SNP locus screening principle comprises the following steps:
s1, determining phenotype values of main economic characters such as growth, feed efficiency, organism composition, meat production, reproduction and the like of 2074 Hu sheep, and identifying genotypes of genome genetic variation of corresponding individuals by utilizing a genome resequencing technology; specifically, the main economic traits measured are mainly 5 categories, namely growth traits, carcass traits, body composition, muscle quality and others, including 59 traits and 199 indexes. Wherein,
the body weight includes: birth weight, weaning weight (56 d); 80d, 100d, 120d, 140d, 160d, and 180d body weight; daily gain includes: 80-100d, 80-120d, 80-140d, 80-160d, 80-180d, 100-120d, 100-140d, 100-160d, 100-180d, 120-140d, 120-160d, 120-180d, 140-160d, 140-180d, and 160-180 d;
Body ruler (body height, body length, chest circumference, tube circumference, tail length, tail width, scrotum circumference) includes: 0d, 100d, 120d, 140d, 160d and 180d, body length, chest circumference, tube circumference; 180d tail length and tail width; 100d, 120d, 140d, 160d, and 180 d;
the feed intake includes: 0-100d, 80-120d, 80-140d, 80-160d, 80-180d, 100-120d, 100-140d, 100-160d, 100-180d, 120-140d, 120-160d,
Daily feed intake of 120-180d, 140-160d, 140-180d and 160-180 d;
the feed conversion rate comprises the following steps: 80-100d, 80-120d, 80-140d, 80-160d, 80-180d, 100-120d, 100-140d, 100-160d, 100-180d, 120-140d, 120-160d, 120-180d, 140-160d, 140-180d, and 160-180 d;
the residual feed intake includes: 80-100d, 80-120d, 80-140d, 80-160d, 80-180d, 100-120d, 100-140d, 100-160d, 100-180d, 120-140d, 120-160d, 120-180d, 140-160d, 140-180d and 160-180 d;
metabolic body weight includes: 80-100d, 80-120d, 80-140d, 80-160d, 80-180d, 100-120d, 100-140d, 100-160d, 100-180d, 120-140d, 120-160d, 120-180d, 140-160d, 140-180d, and 160-180 d;
also pre-slaughter weight, carcass weight, slaughter rate, GR value, eye muscle area, backfat thickness, rib count, chest circumference, carcass length, buttock circumference, tibia circumference head, hoof, coat absolute weight and relative weight; the weight of the cores totaled 250 personality indexes.
S2, recording the phenotype group obtained in the step S1, and finishing phenotype, pedigree and fixed effect files;
s3, estimating breeding values of the phenotypes, the pedigrees and the fixed effect relations which are arranged in the step S2, and calculating a corrected phenotype;
s4, quality control is carried out on the resequenced data obtained in the step S1, and quality control standards are as follows: removing SNP loci with SNP detection rate <30%, minimum allele frequency <0.05, minimum average sequencing depth <5 and no chromosome position information;
s5, selecting the corrected phenotype and the resequenced data with resequenced individuals from the resequenced data obtained in the step S4, and performing whole genome association analysis (GWAS);
performing genome-wide correlation analysis on 250 individual indexes such as body weight, body size, growth speed, feed intake, residual feed intake, feed conversion rate, slaughter rate, eye muscle area, rib number, caudal vertebra number, lumbar vertebra number, GR value, backfat thickness, body composition, visceral fat deposition, omentum fat deposition, caudal fat deposition, inside and outside tail length, inside and outside tail width, caudal fat perimeter, flesh color, intramuscular protein, intramuscular salinity, intramuscular moisture, intramuscular collagen, intramuscular fat, cooked meat rate, drip loss, water loss rate, scrotum perimeter, penis length, penis perimeter, penis weight, blood physiological and biochemical indexes by using a Bonferroni method, and setting a statistical test threshold with remarkable genome level to judge remarkable SNPs (social network points) associated with each character;
Finally, selecting SNP loci with the top 0.00001% of each character significance rank as candidate SNP loci which are obviously related to the characters;
s6, selecting correction phenotype and resequencing data of resequencing individuals from the resequencing data obtained in the step S4, estimating the effect value of the SNP marker by using a rrBLUP method on a reference number group (n=2074), and respectively detecting and explaining SNP loci with larger proportion of 250 personality additive genetic variances; calculating to obtain the effect value of the SNP marker of the corresponding character, and selecting SNP loci with the interpretation additive genetic variance proportion ranking of 0.00001% as candidate loci;
s7, carrying out sliding window screening on the re-sequenced data after quality control obtained in the step S4; sliding a window of 25000Kb length on the chromosome using the oar_rambouille_v1.0 sheep genome assembly result, and screening the window for informative SNP sites with highest MAF, which can be evenly spaced on the genome;
specific screening criteria are as follows: 1) Selecting the SNP locus with the highest MAF in a 25000Kb sliding window; 2) SNP loci with 80% detection rate to ensure stable reproducibility of loci;
finally, in order to avoid the lack of flanking marker information at the beginning/end of each chromosome, the first and last SNPs of each chromosome must be selected; thus, for each constant stain, the highest MAF SNP site in each 25000Kb window was selected and the SNP at the beginning and end of each chromosome was included;
S8, carrying out re-sequencing on the quality-controlled data obtained in the step S4, downloading an oar_rambouille_v1.0 sheep genome annotation file from an Ensemble database, and annotating high-quality SNP loci into corresponding gene areas according to the physical positions of the markers, wherein the genes comprise protein coding genes, non-protein coding genes and partial pseudogenes; meanwhile, regulatory elements such as a Hu sheep genome promoter, an enhancer, a chromatin opening region and the like and an active region are identified based on technologies such as ChIP-seq, ATAC-seq, CUT & Tag and the like; for each gene region, genome promoter, enhancer, chromatin opening region and other regulatory elements and active region, the SNPs annotated to that region will form a single set of SNPs, and the SNP site with the highest MAF is selected from the set;
s9, screening specific loci among varieties: respectively calculating group fixed indexes (Fst) between Hu sheep and other sheep breeds in the whole genome range by utilizing the collected multi-platform genotyping data of the 32 sheep breeds resource materials collected worldwide, and selecting the window of the first 1 permillage of the highest Fst in the whole genome range to take intersection as a significantly selected region;
s10, important SNP loci which are reported by an OMIA database (https:// www.omia.org/home /) and an Ishaep QTLdb database and related to sheep economic traits;
S11, combining SNP loci obtained by screening in each of the steps 5, 6, 7, 8, 9 and 10 (removing repeated screening loci) to form a sheep 1K liquid phase chip SNP set for the design research of a Hu sheep whole genome selection chip;
s12, calculating intervals, linkage disequilibrium and minimum allele frequency between adjacent SNP loci of the functional SNP locus chips, and generating a final sheep 1K liquid phase chip locus set after eliminating abnormal loci, wherein the SNP loci are shown in the following table from No.1 to No. 1088;
TABLE 1SNP loci
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Wherein the first digit in each cell in the table indicates the chromosome number, the last digit indicates the position on the chromosome, and the last letter indicates the base of the reference gene for the locus.
S13, analyzing and selecting 1 DNA region positioned on sheep Y chromosome for sex identification, wherein the sequence is shown as SEQ ID NO. 1: TGTGTTTTCTTTTAATGGAACAGTTTTTATG TCAAAGTTTTGTTGTATCTGAGATTGACCATTAAATATGTATTAGTATATATTAGCATTCTGAAAGTCGTCAGCATATATCCAGTTCAGTTATTAACTACTGGAATACCTACATAGATTTTTCAGCTGTTTTTTAAGCCTAAGTGGAGAAGCAGGGAGAGTGATTAGCATGGATTCGGTATCCTGTGTATTCAGCTATATAACAGGATCGGGCTGTTTTTCATCTTTTTTTTTAAATATAACATTCTCTTTCATTCATAACCATAAACAATTTGTTAGTATGTCTGCTGCACCTTCATCCTTTAAATAAGTATATGAAAATAACTTCACAATGACACCGGTTGTATTTTTAAGCAGGTATTAGCGCCTTCACAAATTCTGATTAGATGTAAACAAAGAAGAAAGCAGAGCGTTAATATCCTGTTAAGCACCTTTGGTGGGTTTGGGCTGGCTGCCAGGAGGTATTGAGGGGAGGTATTGGGGGCGGAGAAATAAATATTTCACTGCAAATTTTGCACCAAGTCAGTCTCTGGTAAGAACAACTTATGAATAGAACGGTGCAATCGTA; the probe designed for the sequence is a nucleotide sequence shown as SEQ ID NO.2-SEQ ID NO.9, and the 5' end of the probe is modified by a biotin group; wherein the sequence is as follows
SEQ ID NO.2：TCCAGTTCAGTTATTAACTACTGGAATACCTACAT AGATTTTTCAGCTGTTTTTTAAGCCTAAGTGGAGAAGCAGGGAGAGTG ATTAGCATGGATTCGGTATCCTGTGTA；
SEQ ID NO.3：TCTCTTTCATTCATAACCATAAACAATTTGTTAGTA TGTCTGCTGCACCTTCATCCTTTAAATAAGTATATGAAAATAACTTCAC AATGACACCGGTTGTATTTTTAAGC；
SEQ ID NO.4：AAGCCTAAGTGGAGAAGCAGGGAGAGTGATTAGC ATGGATTCGGTATCCTGTGTATTCAGCTATATAACAGGATCGGGCTGT TTTTCATCTTTTTTTTTAAATATAACAT；
SEQ ID NO.5：TTCAGCTATATAACAGGATCGGGCTGTTTTTCATCT TTTTTTTTAAATATAACATTCTCTTTCATTCATAACCATAAACAATTTGT TAGTATGTCTGCTGCACCTTCATC；
SEQ ID NO.6：CTTTAAATAAGTATATGAAAATAACTTCACAATGA CACCGGTTGTATTTTTAAGCAGGTATTAGCGCCTTCACAAATTCTGATT AGATGTAAACAAAGAAGAAAGCAGAG；
SEQ ID NO.7：AGGTATTAGCGCCTTCACAAATTCTGATTAGATGT AAACAAAGAAGAAAGCAGAGCGTTAATATCCTGTTAAGCACCTTTGGT GGGTTTGGGCTGGCTGCCAGGAGGTAT；
SEQ ID NO.8：GAGGTATTGAGGGGAGGTATTGGGGGCGGAGAAA TAAATATTTCACTGCAAATTTTGCACCAAGTCAGTCTCTGGTAAGAAC AACTTATGAATAGAACGGTGCAATCGTA；
SEQ ID NO.9：CGTTAATATCCTGTTAAGCACCTTTGGTGGGTTTGG GCTGGCTGCCAGGAGGTATTGAGGGGAGGTATTGGGGGCGGAGAAAT AAATATTTCACTGCAAATTTTGCACCA。
The coverable region of 1088 SNP loci screened in step 12 (i.e., the upstream and downstream sequences covering the locus) should meet the design principle of sheep 1K probe at the same time; the design principle of the sheep 1K probe is as follows: the length of the probe is 110bp, the GC content of the probe is between 30% and 70%, the number of homologous regions is less than or equal to 5, and the selected region does not contain an SSRX region and a GAP region to the maximum extent; designing two nucleotide sequences which have 60-70% of overlapping and cover the SNP locus according to the SNP locus obtained by screening; synthesizing single-stranded nucleotide according to the designed nucleic acid sequence, wherein the synthesized two pieces of the single-stranded nucleotide have the length of 110bp; the DNA nucleotide sequence modified by biotin group at the 5' end is called sheep 1K probe; mixing the two synthesized sheep 1K probes in an equimolar mass manner, and utilizing EDTA and Tris-HC1 mixed solution to fix the volume of the sheep 1K probe mixed solution with the volume of 3 pmol/mL; and (3) packaging the sheep 1K probe mixed solution and the hybridization capture reagent respectively and independently to obtain the sheep 1K liquid phase chip based on targeted capture sequencing.
In this example, the hybridization capture reagent is a GenoBaits DNAseq Library Prep kit from Shijia Boruidi biotechnology Co., ltd, comprising GenoBaits Block I, genoBaits Block II, genoBais 2×Hyb Buffer, genoBaits Hyb Buffer Enhancer, genoBais 2× Beads Wash Buffer, genoBais 10×Wash Buffer I, genoBais 10×Wash Buffer II, genoBais 10×Wash Buffer III, and GenoBais 10× Stringent Wash Buffer packaged independently.
In this embodiment, the sheep reference genome is oar_rambouillet version 1.0.
In this example, the design of sheep 1K probe was performed for 1088 SNP sites and 1Y chromosome DNA region, and the obtained SNP covered by sheep 1K liquid phase chip is shown in fig. 1, which shows that the SNP marker screened by sheep 1K liquid phase chip of the invention covers each autosome, and is basically designed according to the chip design principle, without genome definition gap region.
Step 2, constructing a sheep DNA high-throughput sequencing library by utilizing blood genome DNA of the sheep to be tested;
1) Extracting blood genome DNA of sheep to be tested:
A. 276 Hu sheep are selected from 2 nationally-scaled sheep farms, and blood of corresponding individuals is collected for extracting genome DNA. The sample DNA is extracted by adopting a blood DNA rapid extraction kit (magnetic bead method) developed by Shijia Boruidi biotechnology Co.
B. The DNA concentration of the test sample was determined by Qubit Fluorometric Quantitation (Thermo Fisher) and the integrity of the DNA was checked by 1% agarose gel electrophoresis. And placing the sample which is qualified in detection into a refrigerator at the temperature of 4 ℃ for storage and standby.
2) Constructing a sheep DNA high-throughput sequencing library:
a. and (3) placing 12 mu L of DNA qualified in quality inspection in a 0.2 mu L PCR tube, placing the tube in an ultrasonic breaker to randomly and physically break the DNA, and breaking the fragments to 200-400 bp.
b. To the tube were added 4. Mu. L GenoBaits End Repair Buffer (GenoBaits, shi Jia Boruidi Biotechnology Co., ltd.) and 2.7. Mu. L GenoBaits End Repair Enzyme, and water was added to 20. Mu.L, and the mixture was incubated in an ABI9700 PCR apparatus at 37℃for 20 minutes to complete the end repair and addition of the fragment.
c. The vials were removed from the PCR apparatus, 2. Mu. L GenoBaits Ultra DNA ligase, 8. Mu. L GenoBaits Ultra DNA Ligase Buffer and 2. Mu. L GenoBaits Adapter were added, ultrapure water was added to 40. Mu.L, and then placed on an ABI9700 PCR apparatus for reaction at 22℃for 30 minutes to complete the ligation of sequencing adaptors.
d. 48. Mu.L of Beackman AMPure XP Beads (Beackman Co.) was added to the ligation product to purify the ligation product, and after purification, fragment screening was performed using magnetic beads, retaining the ligation product with an insert of 200-300 bp.
e. mu.L of sequencing adapter with Barcode sequence, 1 mu L P adapter, 10 mu L GenoBaits PCR Master Mix are added into the PCR tube of the previous step, and the mixture is supplemented to 20 mu L by pure water; amplification was performed using an ABI9700 PCR instrument, the amplification procedure being: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 60℃for 30s, and extension at 72℃for 30s; repeating the steps 2-4 for 8 cycles; extending at 72℃for 5min. Different Barcode was used to distinguish between different samples.
f. To the second round of PCR products, 24. Mu. L Beckmen AMPure XP Beads (Beackman Co.) was added, and after pipetting up and down well, 0.2. Mu.L of PCR tubes were placed on a magnetic rack until the solution was clear, the supernatant was discarded and the beads were washed once with 75% ethanol, and the library DNA was eluted with Tris-HCl pH 8.0.
Step 3, mixing the sheep 1K liquid phase chip prepared in the step 1 with the sheep DNA high-throughput sequencing library constructed in the step 2, and capturing DNA fragments containing target sites in the sheep DNA high-throughput sequencing library;
1) DNA hybridization
500ng of the constructed genomic DNA sequencing library was taken, 5 mu L GenoBaits Block I and 2 mu L GenoBaits Block II were added, and the mixture was placed on a Eppendorf Concentrator plus (Eppendorf company) vacuum concentrator and evaporated to a dry powder at a temperature of not more than 70 ℃. 8.5 mu L of GenoBaits 2 XHyb Buffer, 2.7 mu L GenoBaits Hyb Buffer Enhancer and 2.8 mu L of nucleic-Free Water are added into a dry powder tube, the mixture is sucked and beaten by a pipettor, and then placed on an ABI 9700PCR instrument for incubation for 10 minutes at 95 ℃, 3 mu L of synthesized probe (the concentration of the probe is 60 ng/. Mu.L) is taken out of the PCR tube, and the mixture is placed on the ABI 9700PCR instrument for incubation for 2 hours at 65 ℃ after vortex shaking and mixing, so that the probe hybridization reaction is completed.
2) DNA capture
100 mu L GenoBaits DNA Probe Beads (streptavidin covered magnetic beads) is added into the reaction system of the hybridization completion of the previous step, the mixture is sucked up and down for 10 times, and the mixture is placed on an ABI 9700PCR instrument for incubation at 65 ℃ for 45 minutes, so that the magnetic beads are combined with the probes. The probe-bound beads were subjected to a 65℃heat wash with 100. Mu. L GenoBaits Wash Buffer I and 150. Mu. L GenoBaits Wash BufferII, respectively, followed by a 100. Mu. L GenoBaits Wash Buffer I and 150. Mu. L GenoBaits Wash Buffer II wash at room temperature (and 150. Mu. L GenoBaits Wash Buffer III wash, respectively), and the washed beads were resuspended in 20. Mu.L of nucleic-Free Water.
13 μl of resuspended DNA (with magnetic beads) was added to a new 0.2mL PCR tube, and then 15 μ L GenoBaits PCR Master Mix, 2 μ L GenoBaits Primer Mix were added to configure a post-PCR system, and library amplification was performed using an ABI 9700PCR apparatus with the following amplification procedure: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 60℃for 30s, and extension at 72℃for 30s; repeating the steps 2-4 for 15 cycles; extending at 72℃for 5min.
To the post-PCR product was added 45. Mu. L Beckmen AMPure XP Beads (Beackman Co.) and blotted up and down with a pipette, then a 0.2mL PCR tube was placed on a magnetic rack until the solution was clear, the supernatant was discarded and the beads were washed twice with 75% ethanol, and the library DNA was eluted with Tris-HCl pH 8.0. The hybridization capture work of the probe is completed.
3) DNA hybridization capture library quality inspection
The DNA concentration of the library was determined using Qubit Fluorometric Quantitation (Thermo Fisher) and then agarose gel electrophoresis was used to determine if the fragment size of the library DNA was between 300 and 400 bp.
And 4, amplifying and purifying the DNA fragment obtained in the step 3, and after the product is subjected to high-throughput sequencing (MGISEQ 2000 sequencer, china), replying to a sheep reference genome by using a sequencing result for comparison, wherein the sheep reference genome is the Oar_rambouille 1.0 version, and processing the sheep reference genome by using a SAMTOOLS, BWA, GATK and BaseNumberNGS software platform to obtain the genome genotyping of the sheep to be detected.
Example 2 genotyping quality evaluation of sheep 1K liquid phase chip obtained in example 1
SNP detection rate is an important index for measuring the quality of chips, and is generally measured by the loci of autosomes and X chromosomes. Based on the result of typing as in example 1, the detection rate of each SNP site was calculated. The calculation method comprises the following steps: for the ratio of the number of samples successfully detected at a certain SNP locus to the number of all samples, finally, the average value of all SNP detection rates is calculated to obtain the value, the SNP detection rate of the sheep 1K liquid phase chip of the example 1 is very high, the SNP average detection rate is 99.21%, and the genotype detection quality of the sheep 1K liquid phase chip of the example 1 is very good.
Example 3 application of sheep 1K liquid phase chip in sheep whole genome selection
The accuracy of estimated genomic breeding values using 1K chips was assessed using four methods, rrBLUP (VanRaden, 2008), bayesA (Meuwissen et al, 2001), bayesB (Meuwissen et al, 2001), and BayesC (Habier et al, 2011). All individuals of the large-scale sheep population described above were randomly divided into 5 independent subsets. Each subset in turn serves as a validation population and assumes that its phenotype is unknown, while the remaining four subsets serve as reference populations. This process was randomly repeated 10 times. Finally, the arithmetic square root of the spearman correlation coefficient between the corrected phenotype and the Genome Estimated Breeding Value (GEBV) divided by the corresponding trait's genetic transmission is used as an evaluation index for the accuracy of the GEBV. The results indicate that the accuracy of the GEBV for the 33 traits calculated using the low density SNP set is between 0.10 and 0.33, as shown in Table 2.
TABLE 2 estimation accuracy of GEBV estimation in each trait for SNP set for estimating Density using rrBLUP, bayesA, bayesB and BayesC methods
The results show that on the basis of the designed chip, the whole genome selection work can be performed in combination with targeted capture sequencing (more SNPs than the marker loci can be obtained without filling). Sheep breeding work can be carried out at lower cost than sheep Illumina OvineSNP50 solid phase chip.
Example 4 application of sheep 1K liquid chip in sex determination
Blood samples of 30 sheep of known gender were randomly collected from a large-scale sheep farm (collected at 2022, 7 months in the Changxing Yongsheng pasture, inc. of Changcheng county, zhejiang province), 15 male and female animals, together with all sheep of example 1, were subjected to capturing of the target DNA fragment as shown in SEQ ID NO.1 using sheep 1K liquid phase chip. Judging the sex of sheep to be detected according to the presence or absence of the DNA fragment, specifically carrying out liquid phase hybridization on a probe with a nucleotide sequence shown as SEQ ID NO.2-SEQ ID NO.9 and genome DNA, capturing and enriching a target region sequence, then carrying out high-throughput sequencing through a sequencing platform, comparing with SEQ ID NO.1, and if the fragment exists, determining that the sheep to be detected is male, otherwise, determining that the sheep to be detected is female. The final detection result was judged to be 100% accurate.
Claims (10)
1. The preparation method of the sheep 1K liquid phase chip based on targeted capture sequencing is characterized by comprising the following steps:
(1) Screening nucleotide sequences designed and synthesized by SNP loci as probes; the 5' end of the probe is modified by biotin groups, and the modified probe is called a sheep 1K probe;
(2) Mixing the synthesized sheep 1K probe with equal molar mass, and forming sheep 1K probe mixed solution by using mixed solution of EDTA and Tris-HC 1;
(3) And packaging the sheep 1K probe mixed solution and the hybridization capture reagent respectively and independently to obtain the sheep 1K liquid phase chip based on targeted capture sequencing.
2. The method according to claim 1, wherein the principle of screening SNP sites comprises the steps of:
step S1, determining the phenotype value of the main economic character of sheep, and identifying the genotype of the genomic genetic variation of the corresponding individual by utilizing a genomic resequencing technology;
s2, arranging phenotype, pedigree and fixed effect files;
s3, estimating a breeding value by using the phenotype, the pedigree and the fixed effect, and calculating a corrected phenotype;
s4, quality control is carried out on the re-sequencing data, wherein the quality control standard is that SNP loci with SNP detection rate less than 30%, minimum allele frequency less than 0.05, minimum average sequencing depth less than 5 and no chromosome position information are removed;
s5, selecting the corrected phenotype and the resequencing data of the resequencing individual to carry out whole genome association analysis; carrying out whole genome association analysis on the main character indexes by utilizing a mixed linear model, setting a statistical test threshold value of genome level significance by using a Bonferroni method, and judging significant SNPs associated with each character; finally, selecting SNP loci with the top 0.00001% of each character significance rank as candidate SNP loci which are obviously related to the characters;
S6, selecting the corrected phenotype and the resequenced data of the resequenced individuals from the resequenced data obtained in the step S4, estimating the effect value of the reference number group on the SNP marker by using a rrBLUP method, and respectively detecting and explaining the SNP loci with larger proportion of the additive genetic variance of the main economic characters; calculating to obtain the effect value of the SNP marker of the corresponding character, and selecting SNP loci with the interpretation additive genetic variance proportion ranking of 0.00001% as candidate loci;
step S7, sliding window screening is carried out on the re-sequenced data after quality control obtained in the step S4; sliding a window with 25000Kb length on an autosome by using the oar_rambouille_v1.0 sheep genome assembly result, and screening information SNP loci with the highest MAF in the window, wherein the SNP loci can be uniformly distributed at intervals on the genome; s8, carrying out re-sequencing on the quality-controlled data obtained in the step S4, downloading an oar_rambouille_v1.0 sheep genome annotation file from an Ensemble database, and annotating high-quality SNP loci to corresponding gene areas according to the physical positions of the markers, wherein the genes comprise protein coding genes, non-protein coding genes and partial pseudogenes; meanwhile, identifying a Hu sheep genome promoter, an enhancer, a chromatin opening region regulatory element and an active region based on technologies such as ChIP-seq, ATAC-seq, CUT & Tag and the like; for each gene region, genome promoter, enhancer, chromatin opening region and other regulatory elements and active region, the SNPs annotated to that region will form a single set of SNPs, and the SNP site with the highest MAF is selected from the set;
Step S9, screening specific loci among varieties: respectively calculating group fixed indexes (Fst) between Hu sheep and sheep of other varieties in a whole genome range by utilizing the collected multi-platform genotyping data of sheep variety resource materials, and selecting a window of the first 1 permillage of the highest Fst in the whole genome range to take intersection as a significantly selected region;
step S10, important SNP loci which are reported by OMIA database https:// www.omia.org/home/Ishaep QTLdb database and related to sheep economic traits;
step S11, combining SNP loci screened by the four methods together, namely removing repeated screening loci to form a sheep 1K liquid phase chip SNP set;
step S12, calculating the interval, linkage disequilibrium and minimum allele frequency between adjacent SNP loci of the functional SNP locus chips, and generating a final sheep 1K liquid phase chip SNP locus set after eliminating abnormal loci, wherein the SNP loci are shown in a table 1;
TABLE 1 chromosome number and position of SNP site and reference base of the site
Step S13, selecting a sequence design probe with 1 DNA region shown in SEQ ID NO.1 positioned on sheep Y chromosome for sex identification;
the coverage area of the screened SNP locus simultaneously meets the design principle of sheep 1K probes.
3. The preparation method according to claim 2, wherein the design principle of the sheep 1K probe is that the probe length is 110bp, the GC content of the probe is 30% -70%, the number of homologous regions is less than or equal to 5, and the selected region does not contain an SSRX region and a GAP region to the maximum extent; designing two nucleotide sequences which have 60-70% overlap and cover the SNP locus according to the SNP locus obtained by screening; synthesizing single-stranded nucleotide of the designed nucleic acid sequence, wherein the synthesized two nucleic acid sequences are 110bp in length; the DNA nucleotide sequence modified by biotin group at the 5' end is called sheep 1K probe.
4. The preparation method according to claim 2, wherein the probe for sex determination is shown in SEQ ID NO.2-SEQ ID NO.9, and the 5' end of the probe is modified with a biotin group.
5. The method of claim 1, wherein the hybridization capture reagent comprises an individually packaged GenoBaits DNAseq Library Prep kit, specifically comprising individually packaged GenoBaits Block I, genoBaits Block II, genoBaits 2 x Hyb Buffer, genoBaits Hyb Buffer Enhancer, genoBaits 2 x Beads Wash Buffer, genoBaits 10 x Wash Buffer I, genoBaits 10 x Wash Buffer ii, genoBaits 10 x Wash Buffer III, and GenoBaits 10 x Stringent Wash Buffer.
6. A method for targeted capture sequencing-based genotyping of sheep genome, comprising the steps of:
step 1, preparing a sheep 1K liquid phase chip based on targeted capture sequencing by adopting the preparation method of claim 1;
step 2, constructing a sheep DNA high-throughput sequencing library by utilizing genome DNA of sheep to be detected;
step 3, mixing the sheep 1K liquid phase chip prepared in the step 1 with the sheep DNA high-throughput sequencing library constructed in the step 2, and capturing DNA fragments containing target sites in the sheep DNA high-throughput sequencing library;
and 4, amplifying and purifying the DNA fragment obtained in the step 3, and after high-throughput sequencing, replying the product to a sheep reference genome by using a sequencing result for comparison, thereby obtaining the genome genotyping of the sheep to be tested.
7. The method according to claim 6, wherein in the step 2, the method for constructing the sheep DNA high-throughput sequencing library is that the genome DNA of the sheep to be tested is fragmented by adopting an ultrasonic breaking method, the tail ends of the fragmented fragments are subjected to filling-up repair and A tail addition, the sequencing joint is connected, the connection products are purified, the fragment screening is carried out by using magnetic beads, the connection products of 200-300 bp of the inserted fragments are reserved, and then the sequencing joint with the Barcode sequence and the P5 joint are used for PCR amplification, so that the sheep DNA high-throughput sequencing library is obtained.
8. The method according to claim 6, wherein in step 3, the sheep 1K probe and the streptavidin-coated magnetic beads are mixed, the target sheep genomic DNA fragment is adsorbed to the magnetic beads by binding biotin and streptavidin, and then the DNA fragment in the non-target region is washed out by elution treatment, thereby obtaining the desired sheep genomic DNA fragment.
9. The method of claim 6, wherein in step 4, high throughput sequencing data is processed through SAMTOOLS, BWA, GATK and basenumber ngs software platforms to obtain SNP genotyping at a specific site in an individual.
10. Use of the sheep 1K liquid phase chip obtained by the preparation method of claims 1-5 in sheep molecular breeding.
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