Identification Of SNPs In Water Buffalo Using Next Generation Sequencing Data

Illumina next generation sequencing data from 48 water buffalo, representing the Mediterranian, Murrah, Jaffrabadi, and Nilli-Ravi breeds, were used to identify SNPs. The iAnimal cyberinfrastructure system was used to facilitate all sequence analyses. Short sequences were aligned to the Bos taurus genome (UMD3.1) using BWA and SNPs were called using GATK. The identified SNPs were sorted into homozygous and heterozygous categories. Only SNPs that were heterozygous in at least one individual within a breed were retained to prevent the identification of species specific SNPs (i.e. fixed/homozygous in buffalo compared to Bos taurus). Each individual was genotyped from their original SNP calls using the unique list of SNPs obtained from the previous step. The minor allele frequency was calculated within breed and across breeds. Filtering was conducted using a series of perl, R, and shell scripts. The FastaAlternateReference function of GATK was used to create a “buffalo corrected” version of UMD3.1, identical to the bovine genome, except for targeted changes to loci where homozygous SNPs were identified across all individuals when compared to the bovine genome. These SNPs were modified in the bovine genome to reflect the true buffalo sequence in the corrected reference. Flanking sequence was retrieved from the buffalo corrected reference to create probes for all filtered SNPs. We identified a total of 22,293,567 SNPs meeting the filtering criterion across all breeds, including 17,096,560 (Mediteranian), 7,847,323 (Murrah), 9,121,702 (Jaffrabadi) and 11,484,032 (Nilli-Ravi) unique SNPs across breeds.