Fig. 2

Candidate aberrant splicing events detected with RNA-seq analysis of undiagnosed cases. (a) Quality check of skeletal muscle (SkM) samples by principal component analysis (PCA) of gene expression level. Six undiagnosed myopathy of nonspecific muscle pathology (NP), five Duchenne muscular dystrophy (DMD), two Ullrich congenital muscular dystrophy (UCMD), two genetically diagnosed NM with biallelic truncating NEB variants (NM-D), and six undiagnosed NM (NM-U) cases were analyzed. (b,c) Sashimi plots of RNA-seq reads in NM-U cases: (b) NM-U1 and (c) NM-U2, NM-U3, and NM-U4 . Upper black plot: an NP case; lower red plots: NM-U cases. Variants identified by Sanger sequencing are indicated below the plots. Exonic regions are indicated as solid horizontal lines above the plots. Exon and intron sizes are arbitrary for clear visualization of splicing events. Sample names are described in the upper right or left. The read counts spanning each splicing junction are described on or nearby the corresponding arcs. Arcs indicate splicing junctions, and arcs with asterisks indicate candidate aberrant splicing events after narrowing down the list of candidates based on their ψ-D, ψ-FC, and p values. Numbers on arcs describe the read counts supporting the splicing junctions. Arrows indicate partial intron 175 retention. X- and y-axes indicate corresponding genomic region, and reads per kilobase million (RPKM), respectively. The right-hand plots are an enlarged selection of the left-hand plots in (c). (d) Integrative Genomics Viewer (IGV) image of subsampled RNA-seq reads of the 11-bp region around the c.24684G>C variant in NM-U2. The pink lines indicate each RNA-seq read mapped to the targeted region in the gray background. Reads restricted to exon 175 indicate splicing events excising intron 175, while reads spanning exon 175 and intron 175 indicate intron 175 retention. Exon 175 and intron 175 regions are shown as horizontal solid and dotted lines on top. The c.24684 base is indicated at the bottom. The c.24684G>C variant in each read is colored brown (line). Note that most of the spliced reads did not contain c.24684G>C, while most of the reads retaining intron 175 did contain c.24684G>C. Except for the third base, the first five bases of intron 175 base (c.24684+1 to +5) were the same as those of exon 176 (c.24685 to 24689), implying that reads in line with the dashed vertical line could be erroneously mapped. Differently colored sites in the pink line: bases not matched to the reference sequence.