50k liquid-phase chip for pigs based on multiple single nucleotide polymorphisms

A method for SNP marker selection and probe preparation for a 50K liquid phase chip used for multiple single nucleotide-polymorphism, which utilizes whole-genome sequencing data of pig breeds to mine and screen target SNP loci, then designs and optimizes probes for the target SNP loci, ultimately resulting in the determination of the probes;

• wherein the method includes the following steps:
• Step 1, determining target site SNPs:
• based on whole-genome sequencing data of three pig breeds—Duroc, Large White, and Landrace—and aligning them to the whole-genome sequencing data of pig breeds, genomic regions with moderate linkage disequilibrium between markers were selected, and targeted capture sites were screened, i.e., target site SNPs;
• Step 2, designing probes based on the determined target site SNPs:
• utilize the multiple single nucleotide polymorphism technologies to design 1-4 probes, each 110 bp in length, centered on each target SNP;
• each probe covers the target SNP, with a total probe coverage of 165 bp around the target SNP;
• wherein the principles for probe design are:
• 1) select probes with a content between 30% and 80%;
• 2) choose regions with a number of homologous areas ≤5;
• 3) select probe areas that do not contain SSR, N regions;
• and
• Step 3, selecting and optimizing probes containing high-quality mSNPs:
• probes are hybridized and sequenced, and the genotyping quality of mSNPs, including target sites, is detected;
• set a missing rate of NA<0.1, a minimum allele frequency (MAF) ≥0.05, and heterozygosity (Het) <0.5 as standards to screen mSNPs, removing those that do not meet the standards;
• if the probe does not meet the genotype quality control requirements of mSNPs, delete the probe and the corresponding target site SNP, redesign new probes according to Steps 1 and 2, and continue to test and optimize the probes as per this step;
• wherein the mSNPs that meet the quality inspection requirements are finally used as the target sites of the 50K mSNP liquid-phase chip.