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


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.

Authors: Laura Schulz, Manuel Torres-Diz, Mariela Cortés-López, Katharina E. Hayer, Mukta Asnani, Sarah K. Tasian, Yoseph Barash, Elena Sotillo, Kathi Zarnack, Julian König, Andrei Thomas-Tikhonenko

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