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

ABSTRACT: 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.

SUBMITTER: Schulz L

PROVIDER: S-EPMC8240250 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies

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