Detecting alternatively spliced transcript isoforms from single-molecule long-read sequences without a reference genome.
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. We applied Iso-Seq™ to investigate AS in Amborella trichopoda, a phylogenetically pivotal species that is sister to all other living angiosperms. Our 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, we 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 novoAS detection pipeline provides a method to accurately characterize and identify bona fide alternatively spliced transcripts in any nonmodel system that lacks a reference genome sequence. Hence, our pipeline has huge potential applications and benefits to the broader biology community.
Department of Biology, University of Florida, Gainesville, FL, 32611-8525, USA.|Florida Museum of Natural History, University of Florida, Gainesville, FL, 32611-7800, USA.
Xiaoxian
http://orcid.org/0000-0001-6386-1527
X
Liu
Department of Biology, University of Florida, Gainesville, FL, 32611-8525, USA.
Wenbin
W
Mei
Florida Museum of Natural History, University of Florida, Gainesville, FL, 32611-7800, USA.|Genetics Institute, University of Florida, Gainesville, FL, 32610, USA.
Pamela S
PS
Soltis
Department of Biology, University of Florida, Gainesville, FL, 32611-8525, USA.|Florida Museum of Natural History, University of Florida, Gainesville, FL, 32611-7800, USA.|Genetics Institute, University of Florida, Gainesville, FL, 32610, USA.
Douglas E
DE
Soltis
Department of Biology, University of Florida, Gainesville, FL, 32611-8525, USA.|Genetics Institute, University of Florida, Gainesville, FL, 32610, USA.
W Brad
WB
Barbazuk
D017398:Alternative Splicing; D020869:Gene Expression Profiling / Q000379:methods*; D017343:Genes, Plant; D019684:Magnoliopsida / Q000235:genetics*; D020033:Protein Isoforms / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics
D020033:Protein Isoforms; D012333:RNA, Messenger

Amborella trichopoda
; alternative splicing; pipeline; single-molecule long-read sequencing; transcriptomics