Micro-RNA Quantification, Target Gene Identification, and Pathway Analysis.

RNA sequencing has become a powerful tool for profiling the expression level of small RNAs from both solid tissues and liquid biopsies. In conjunction with pathway analysis, it offers exciting possibilities for the identification of disease specific biomarkers. In this chapter, we describe a workflow for processing this type of sequencing data. We start by removing technical sequences (adapters) and by performing quality control, a critical task that is necessary to identify possible issues caused by sample preparation and library sequencing. We then describe read alignment and gene-level abundance estimation. Building on these results, we normalize expression profiles and compute differentially expressed microRNAs between sample groups of interest. We conclude by showing how to employ pathway analysis to identify molecular signatures corresponding to biological processes that are significantly altered by the action for microRNAs.