Unsplicing of specific genes because of spliceosomal mutations as detected by deep RNA sequencing. Next-generation–based RNA deep sequencing was used to quantitatively study splicing patterns. (A) The top panel shows the intron 5 and exon 6 boundary of TET2 (dotted line). Five reads correspond to transcripts that were not spliced (unspliced) and 4 were spliced at this boundary. The bottom panel shows read counts at the 5′ and 3′ splice sites of each intron (3-10) of TET2. White and black bars indicate the number of spliced and unspliced reads, respectively. In a case of AML with a U2AF1 mutation, more unspliced than spliced reads were observed at the 3′ splice site of intron 5 (left panel), probably because of a loss of spliceosome function. However, unspliced RNAs were less frequent than spliced RNAs in WT RNA sequencing (right panel). (B) At both the 3′ and 5′ splice sites of RUNX1 intron 6, unspliced reads were more frequent than spliced reads in AML cases with U2AF1 and SRSF2 mutations. However, there were fewer unspliced transcripts at the same site in WT and SF3B1 mutant samples. Splicing abnormalities in the selected genes are summarized (bottom right), including the results presented in detail in supplemental Figures 7 and 8.