Practice question

Distinguishing novel transcripts from annotated ones depends heavily on post-sequencing alignment analysis. After sequencing cDNA libraries, reads are mapped with splice-aware aligners such as STAR or HISAT2 that can split alignments across introns. Reads spanning previously unannotated exon-exon junctions, retained introns or alternative splice sites emerge only during alignment. Reverse transcription and library preparation create cDNA fragments, sequencing depth improves sensitivity, but computational alignment reveals exon connectivity, fusion genes and novel isoforms. Hence alignment is step that identifies transcript novelty and structural variation.