Detecting alternatively spliced transcript isoforms from single-molecule long-read sequences without a reference...

Area of Science:

• Genomics
• Molecular Biology
• Evolutionary Biology

Background:

• Alternative splicing (AS) generates significant transcriptomic and proteomic diversity.
• Studying AS is difficult in species without well-annotated reference genomes.
• Iso-Seq™ data offers advantages for isoform-level transcriptome analysis.

Purpose of the Study:

• To investigate alternative splicing in Amborella trichopoda, a key angiosperm species, using Iso-Seq™.
• To develop and validate a de novo pipeline for AS detection without a reference genome.

Main Methods:

• Application of Iso-Seq™ technology to Amborella trichopoda.
• Reference-based AS detection using Iso-Seq™ data.
• Development of a de novo pipeline for AS isoform detection from Iso-Seq™ data.

Main Results:

• Iso-Seq™ improved recovery of large transcripts, gene locus identification, and gene model correction compared to RNA-Seq.
• Reference-based AS detection identified AS in a higher fraction of multi-exonic genes (45.8%) than RNA-Seq (37.5%).
• The de novo AS detection pipeline achieved a 66%-76% success rate in identifying AS events.

Conclusions:

• Iso-Seq™ is effective for AS detection, particularly in species like Amborella trichopoda.
• The developed de novo pipeline enables accurate AS characterization in non-model systems lacking reference genomes.
• This approach has broad applicability for transcriptomic research in diverse species.