Retained introns in long RNA-seq reads are not reliably detected in sample-matched short reads

Abstract

Abstract Background There is growing interest in retained introns in a variety of disease contexts including cancer and aging. Many software tools have been developed to detect retained introns from short RNA-seq reads, but reliable detection is complicated by overlapping genes and transcripts as well as the presence of unprocessed or partially processed RNAs. Results We compared introns detected by 8 tools using short RNA-seq reads with introns observed in long RNA-seq reads from the same biological specimens. We found significant disagreement among tools (Fleiss’ $$\kappa = 0.113$$ κ = 0.113 ) such that 47.7% of all detected intron retentions were not called by more than one tool. We also observed poor performance of all tools, with none achieving an F1-score greater than 0.26, and qualitatively different behaviors between general-purpose alternative splicing detection tools and tools confined to retained intron detection. Conclusions Short-read tools detect intron retention with poor recall and precision, calling into question the completeness and validity of a large percentage of putatively retained introns called by commonly used methods.