Computational detection and experimental validation of segmental duplications and associated copy number variants in river buffalo (Bubalus bubalis)

Duplicated sequences are the important source of gene innovation and structural variation within mammalian genomes. Using a read depth approach based on next-generation sequencing, we performed a genome-wide analysis of segmental duplications (SDs) and associated copy number variants (CNVs) in water buffalo (Bubalus bubalis). Aligning to the UMD3.1 cattle genome, we estimated 44.6 Mb (~1.73% of cattle genome) segmental duplications in the autosomes and X chromosome using the sequencing reads of Olimpia (the sequenced water buffalo). 70.3% (70/101) duplications were experimentally validated using the fluorescent in situ hybridization. We also detected a total of 1344 CNV regions across 14 additional water buffalos as well as Olimpia, amounting to 59.8Mb of variable sequence or 2.2% of the cattle genome. The CNV regions overlap 1245 genes and are significantly enriched for specific biological functions such as immune response, oxygen transport, sensory system and signalling transduction. Additionally, we performed array Comparative Genomic Hybridization (aCGH) experiments using the 14 water buffalos as test samples and Olimpia as the reference. Using a linear regression model, significant and high Pearson correlations (r = 0.781) were observed between the digital aCGH values and aCGH probe log2 ratios. We further designed Quantitative PCR assays to confirm CNV regions within or near annotated genes and found 74.2% agreement with our CNV predictions. Overall design: Whole genome high-denstiy CGH arrays manufactured by Agilent containing ~974,016 oligonucleotide probes were designed and fabricated on a single slide to provide an evenly distributed coverage on cattle UMD3.1 with an average interval of ~3.1 kb between probes. The reference animal chosen was Olimpia, an Italian Mediterranean river buffalo.