Single molecule long-read sequencing reveals predominantly “all or none” splicing of multi-intron transcripts. (A) Full-length transcripts mapping to nine genes with more than one intron are aligned underneath each gene diagram and color-coded according to their splicing profile. Thin lines in transcripts indicate that the intron sequence is absent (due to splicing). Transcripts overlapping with <2 introns (“others,” gray) cannot be used to analyze the order of intron splicing. The inset shows five transcripts of gene SPBC428.01c, highlighting two subclasses of partially spliced transcripts and that transcript 3′ ends refer to Pol II position. (B) All unspliced, all spliced, and partially spliced fraction in the transcriptome. The large pie chart shows the proportion of the three splicing categories from nRNA LRS. The small pie chart depicts the fraction of partially spliced transcripts, which show intron removal “in order” (all introns are spliced upstream of an unspliced intron in a particular transcript; compare zoom-in of SPBC428.01c in A), “not in order” (at least one intron is unspliced upstream of a spliced intron in a particular transcript), or a mixed pattern (compare inset of SPBC428.01c in A).
