Fig. 3: Detection of mis-splicing caused by variants in PDSS1 by using ONT sequencing.
From: Biallelic variants in coenzyme Q10 biosynthesis pathway genes cause a retinitis pigmentosa phenotype
a IGV visualisation of ONT single molecule sequencing of Individual family 3, II:2. Reads are aligned to the PDSS1 transcript (PDSS1-201 ENST00000376215.10, NM_014317.5) and reads are grouped according to position c.589 A > G. Variant c.722-2 A > G leads to skipping of 222 bp (exon 7–8) or alternate acceptor usage in exon 8 (14 bp downstream). b IGV visualisation of ONT single molecule sequencing of individual from family 5, II:1. Reads are aligned to the human genome (build GRCh38) and PDSS1 exon 6 is shown. Reads are grouped according to position c.589 A > G. Inclusion of the alternate exon (NM_001321979.1) is enriched on the c.468-25 A > G allele (90.25% versus 6.5-10.14% controls). c Schematic representation of cryptic splicing caused by c.722-2 A > G and c.468-25 A > G PDSS1 variants and agarose gel analysis of RT-PCR reactions showing two transcripts in all samples. The 941 bp band corresponds to the canonical transcript and the 1025 bp band corresponds to the alternate exon 6 transcript. PDSS1 NC shows a brighter band for the canonical transcript, while PDSS1 -25 shows a reduced intensity. PDSS1 -2 shows no additional transcripts. PDSS1 NC – normal control, PDSS1 -2 – individual with c.722-2 A > G. PDSS1 -25 – individual with c.468-25 A > G.
