Identification of Novel Splice Sites and Alternative Splicing Events in <italic>Drosophila melanogaster</italic> Using RNA-seq Data

Abstract

Gene structure prediction is the first and most fundamental step to genome analysis and annotation. Splice site and alternative splicing (AS) prediction is particularly challenging for eukaryotes. With the Next Generation sequencing technologies, RNA-seq has been used in identification of splice site and alternative splicing. In this work, 39718 fruit fly splice sites were identified based on <italic>Drosophila melanogaster</italic> testis RNA-seq data by using Tophat software, of which 10584 were new discoveries. By different donor/acceptor splice site combinations, a computational identification method has been developed and applied to predict 8477 alternative splicing events (containing four distinct classes of AS events: alternative donor site, alternative acceptor site, intron retention and exon skipping). RT-PCR successfully validated novel alternative splicing events and new isoforms in two genes. Our result indicates that RNA-seq was not only an effective and accurate method for splice site and AS event detection, but also a new technique for deciphering molecular mechanism of RNA splicing further.