Overview of RNA-Seq data analysis. Following typical RNA-Seq experiments, reads are first aligned to a reference genome. Second, the reads may be assembled into transcripts using reference transcript annotations or de novo assembly approaches. Next, the expression level of each gene is estimated by counting the number of reads that align to each exon or full-length transcript. Downstream analyses with RNA-Seq data include testing for differential expression between samples, detecting allele-specific expression, and identifying expression quantitative trait loci (eQTLs).