Abstract
AbstractAccurate alignment of transcribed RNA to reference genomes is a critical step in the analysis of gene expression, which in turn has broad applications in biomedical research and in the basic sciences. We reveal that widely used splice-aware aligners, such as STAR and HISAT2, can introduce erroneous spliced alignments between repeated sequences, leading to the inclusion of falsely spliced transcripts in RNA-seq experiments. In some cases, the ‘phantom’ introns resulting from these errors make their way into widely-used genome annotation databases. To address this issue, we present EASTR (Emending Alignments of Spliced Transcript Reads), a software tool that detects and removes falsely spliced alignments or transcripts from alignment and annotation files. EASTR improves the accuracy of spliced alignments across diverse species, including human, maize, and Arabidopsis thaliana, by detecting sequence similarity between intron-flanking regions. We demonstrate that applying EASTR before transcript assembly substantially reduces false positive introns, exons, and transcripts, improving the overall accuracy of assembled transcripts. Additionally, we show that EASTR’s application to reference annotation databases can detect and correct likely cases of mis-annotated transcripts.
Funder
U.S. Department of Health & Human Services | National Institutes of Health
National Science Foundation
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary