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.

High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance

“…Read pairs of each minigene were mapped to the respective minigene (including all mutations with penetrance ≥0.8, but excluding insertions and deletions) using STAR 58 (version 2.6.1b). An annotation of three isoforms (exon 2 inclusion and skipping, as well as the artefact PCR product Δex2part which lacks an internal fragment of exon 2 due to a reverse transcription artefact 65 ) was provided to STAR during mapping and an --sjdbOverhang of 259 was set. When running STAR, all SAM attributes were written, up to ten mismatches were allowed, soft-clipping was prohibited on both ends of the reads and only uniquely mapping reads were kept for further analysis.…”

Landscape of exitrons in gastric cancer

“…Second, it is better to identify exitrons using RNA-based, full-length sequencing strategy due to potential artifacts found recently. 49 But this technique is not easily performed. Third, most samples in CPTAC gastric cancer cohort are derived from diffuse-type early-onset gastric cancer patients.…”

ScisorWiz: visualizing differential isoform expression in single-cell long-read data

“…Differential isoform expression between cell types and across conditions plays a major role in the diversification of the proteome ( Nilsen and Graveley, 2010 ) and functionality of transcripts in the cell ( Yang et al , 2016 ). Long-read sequencing has become widely used to address this problem ( Au et al , 2013 ; Bolisetty et al , 2015 ; Koren et al , 2012 ; Leung et al , 2021 ; Oikonomopoulos et al , 2016 ; Ruiz-Reche et al , 2019 ; Schulz et al , 2021 ; Sharon et al , 2013 ; Tilgner et al , 2015 ), and with applications to single-cell isoform sequencing studies ( Arzalluz-Luque et al , 2022 ; Gupta et al , 2018 ; Hardwick et al , 2022 ; Joglekar et al , 2021 ; Volden and Vollmers, 2022 ). These approaches have been reviewed in Hardwick et al (2019) .…”