UPDATE: The study has been published in the journal G3: GENES, GENOMES, GENETICS and featured on its cover.
ORIGINAL POST (March 12, 2018):
A new preprint from scientists at the University of California, Davis, demonstrates the usefulness of SMRT Sequencing and the Iso-Seq method to enable comprehensive transcriptome analysis even in the absence of a reference genome assembly. The team conducted this study on the grape used to make Cabernet Sauvignon.
Lead author Andrea Minio, senior author Dario Cantu, and collaborators used SMRT Sequencing to generate full-length transcripts of Vitis vinifera cv. Cabernet Sauvignon, analyzing RNA collected from four replicates at each of four important stages of ripening. Previously, this team used PacBio sequencing to generate a highly contiguous genome assembly for the wine grape. In this new effort, they focused on characterizing the networks associated with metabolism and berry development.
Cantu and his team are used to studying plants that lack reference-grade genome assemblies. They adopted the Iso-Seq method for this new project as a means of evaluating it for use on other targets without references in the future. “Iso-Seq is an ideal technology for reconstructing a transcriptome without a reference sequence and for resolving isoforms,” they report in the preprint, noting that full-length transcripts capture splice variants and noncoding RNAs. Their wine grape analysis yielded more than 170,000 transcripts associated with 13,402 genes. “Full-length transcripts refined approximately one third of the gene models predicted using several ab initio and evidence-based methods,” the scientists write. “The Iso-Seq information also helped identify 563 additional genes, 4,803 new alternative transcripts, and the 5’ and 3’ UTRs in the majority of predicted genes.”
The results indicated significant changes in gene expression depending on developmental stage. Just a quarter of loci were represented at all four stages tested; a third of loci were detected only at specific stages, “confirming the importance of collecting different stages of development to capture the complexity of the berry transcriptome,” the authors note.
“This study demonstrates that Iso-Seq data can be used to compile a comprehensive reference transcriptome that represents most genes expressed in a tissue undergoing extensive transcriptional reprogramming,” the scientists conclude. “The pipeline described here can be of even greater value for projects aiming to reconstruct the gene space in plant species with complex and large genomes that have not been resolved yet.”