Which step in RNA-seq distinguishes novel transcripts?
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.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.