The results indicated that the percent of concordant SNPs and Indels from Freebayes and GATK after our new method were significantly improved 12%−32% compared with raw variants and advantageously found low frequency variants of individual sheep involved several traits including nipples number (GPC5), scrapie pathology (PAPSS2), seasonal reproduction and litter size (GRM1), coat color (RAB27A), and lentivirus susceptibility (TMEM154). This evidence concerns the gene TMEM154 and scrapie.