Alternative splicing (AS) is a major source of transcript and proteome diversity, but examining AS in species without well-annotated reference genomes remains difficult. Research on both human and mouse has demonstrated the advantages of using Iso-Seq™ data for isoform-level transcriptome analysis, including the study of AS and gene fusion.
Researchers at the University of Florida applied Iso-Seq™ to investigate AS in Amborella trichopoda, a phylogenetically pivotal species that is sister to all other living angiosperms. Their data show that, compared with RNA-Seq data, the Iso-Seq™ platform provides better recovery on large transcripts, new gene locus identification, and gene model correction. Reference-based AS detection with Iso-Seq™ data identifies AS within a higher fraction of multi-exonic genes than observed for published RNA-Seq analysis (45.8% vs. 37.5%). These data demonstrate that the Iso-Seq™ approach is useful for detecting AS events. Using the Iso-Seq-defined transcript collection in Amborella as a reference, the researchers further describe a pipeline for detection of AS isoforms from PacBio Iso-Seq™ without using a reference sequence (de novo). Results using this pipeline show a 66-76% overall success rate in identifying AS events. This de novo AS detection pipeline provides a method to accurately characterize and identify bona fide alternatively spliced transcripts in any non-model system that lacks a reference genome sequence. Hence, this pipeline has huge potential applications and benefits to the broader biology community.
Availability – and code: GitHub: https://github.com/liuxiaoxian/IsoSeq_AS_de_novo