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Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant  CD19  pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within  CD19  exon 2. While “exitrons” are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the  CD19  exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed “falsitrons,” that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.   Supplementary Information  The online version contains supplementary material available at 10.1186/s13059-021-02411-1.

Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts