Identification of IsomiRs vis-à-vis Functional Classification of the Target-Genes Associated with Brucellosis and Paratuberculosis in Indian Water Buffaloes

IsomiRs, the variants of commonly occurring canonical miRNAs, exhibit variation at the 3` or 5` terminus. In the current study, PBMCs were isolated from peripheral blood of four experimental groups of animals: healthy control animals of Murrah breed, Brucellosis-Murrah, Brucellosis NiliRavi breed and Paratubeculosis-Murrah buffaloes (3 to 6 biological replicates, depending on availability of cases). The small RNA (sRNA) was extracted from the pooled-PBMCs (for each of the groups) using miRVana commercial kits and deep-sequenced using Ion-Torrent PGM^™ platform (using two chips viz. 318 chip and 316 chip). The NGS data was processed and further analyzed to identify miRNAs and the isomiRs using miRanalyzer tool. Up and down-regulated isomiRs (ratio >2) of each of the three diseased groups were identified with respect to the healthy group, using R-environment. The uniquely expressed isomiRs that were detected either in the healthy or in one of diseased groups being compared were identified. The target genes of these selected isomiRs were predicted using three different online tools (TargetScan, miRDb, PicTar). The target genes were further used for gene ontology and pathway analysis (Panther classification system) to study their biological as well as functional roles. In total 153, 125 and 139 isomiRs were identified that are common in three diseased groups as compared to the healthy Murrah group. Functional classification of the target genes indicated that these are involved in different systems biology related functions like molecular binding, enzyme modulation, signal modulation etc., however, the specificity of function also varied in the three diseased groups.