Practice question

RNA sequencing analysis begins with mapping reads to a reference genome using splice-aware aligners that handle exon-intron boundaries to identify novel transcripts. This computational step reveals transcript structure, exon connectivity, and novel junctions indicating alternative splicing or gene fusion events absent from annotation databases. Reverse transcription creates complementary DNA copies, while library preparation adds adapters for flow cell binding. Sequencing depth determines sensitivity for low-abundance transcripts, but identification of structural novelty depends critically on alignment patterns. Accurate alignment therefore enables discovery of isoform diversity across conditions.