Fig. 3

HYDIN2 transcript diversity and ORF potential. a Long-range RT-PCR amplicons spanning the first exon identified by 5' RACE to putative terminal exons 19 and 43 (primer pairs shown as half arrows) were targeted for long-read single-molecule sequencing. A total of 12,744 amplicons were characterized and isoform content visualized for each product (row), with exons (columns) colored based on whether they are part of the canonical HYDIN gene structure (blue) or exapted from flanking sequence (green). b HYDIN and HYDIN2 transcript isoforms based on long-read sequencing of RT-PCR products. Exons corresponding to the duplicated segment (blue shading) and flanking sequences for HYDIN (white) and HYDIN2 (green). Three HYDIN2 isoforms were identified (isoforms A, B, and F) and an isoform that spans the segmental duplication on both sides (isoform G) was constructed from multiple overlapping reads. The full-length, canonical HYDIN (ENST00000393567.6) and its shorter isoform (ENST00000321489.9) are shown. Exons in gray are subject to alternative splicing. c Predicted ORFs for HYDIN2 (green bars) are shown with respect to HYDIN gene structure. Coding differences are indicated above exons, numbered with respect to the canonical isoform of the ancestral gene. Circles indicate synonymous (black), non-synonymous (blue), and indel (red) differences. Note: a 2095 bp HYDIN2 deletion eliminates part of the intron 41 and exon 42, including the splice acceptor for exon 42. Exon 42 is skipped and exon 41 is rarely observed in HYDIN2 transcripts. Productive HYDIN2 transcripts are unlikely to continue past exon 42. The three longest ORFs are predicted to be 1852 aa (Isoform F, exons 7-39), 668 aa (Isoform B; exons 7-19) and 467 aa (Isoform A, exons 11-19); only Isoform A lacks multiple exons 5′ to the ORF