Example：10.1021/acsami.1c06204 or Chem. Rev., 2007, 107, 2411-2502
FLAME: long-read bioinformatics tool for comprehensive spliceome characterization RNA (IF4.942), Pub Date : 2021-10-01, DOI: 10.1261/rna.078800.121 Isak Holmqvist, Alan Bäckerholm, Yarong Tian, Guojiang Xie, Kaisa Thorell, Ka-Wei Tang
Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.