Development and comparison of RNA-sequencing pipelines for more accurate SNP identification: practical example of functional SNP detection associated with feed efficiency in Nellore beef cattle

Optimization of an RNA-Sequencing (RNA-Seq) pipeline is critical to maximize power and accuracy to identify genetic variants, including SNPs, which may serve as genetic markers to select for feed efficiency, leading to economic benefits for beef production. This study used RNA-Seq data (GEO Accession ID: PRJEB7696 and PRJEB15314) from muscle and liver tissue, respectively, from 12 Nellore beef steers selected from 585 steers with residual feed intake measures (RFI; n = 6 low-RFI, n = 6 high-RFI). Three RNA-Seq pipelines were compared including multi-sample calling from i) non-merged samples; ii) merged samples by RFI group, iii) merged samples by RFI and tissue group. The RNA-Seq reads were aligned against the UMD3.1 bovine reference genome (release 94) assembly using STAR aligner. Variants were called using BCFtools and variant effect prediction (VeP) and functional annotation (ToppGene) analyses were performed.