UPDATED
February 21, 2018
Congratulations to Winston Timp’s team on the publication of their Iso-Seq analysis of hummingbird! The paper is now available at GigaScience.
ORIGINAL POST
April 4, 2017
A new preprint offers an enticing look at transcriptome results from analysis of a hummingbird using SMRT Sequencing. In this study, scientists found new clues to explain unique attributes of the bird’s metabolism. The work was made possible through full-length isoform sequencing, which allowed deep, assembly-free analysis even though no reference genome was available.
“Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris” is now available on BioRxiv. From Rachael Workman, Alexander Myrka, Elizabeth Tseng, William Wong, Kenneth Welch, and Winston Timp, the paper describes a project designed to better understand how hummingbirds switch metabolic gears to focus on sugars or lipids as needed. “This metabolic flexibility is remarkable both in that the birds can switch between exclusive use of each fuel type within minutes,” they write, “and in that de novo lipogenesis from dietary sugar precursors is the principle way in which fat stores are built, sometimes at exceptionally high rates, such as during the few days prior to a migratory flight.”
The team used the Iso-Seq method with long-read PacBio data to generate full-length isoform sequences, focusing on the liver of Archilochus colubris. According to the paper, this represents “the first high-coverage transcriptome of any single avian tissue.” They also aligned transcripts to Calypte anna, a recently completed hummingbird assembly that also made use of SMRT Sequencing.
Workman et al. report that the use of long-read PacBio data allowed for more accurate views of isoforms and alternative splicing, even without a reference genome. “Using full-length transcript data, we found alignment unnecessary to generate clear pictures of the gene isoforms,” they note. “The long reads negate the need for transcript assembly, a precarious analysis in the absence of a genome.” Nearly half of the reads in the final analysis covered full-length genes, including the 5’ and 3’ ends as well as the polyA tail.
The team used the COGENT pipeline to assign transcripts to gene families and focus on unique isoforms. “COGENT is specifically designed for transcriptome assembly in the absence of a reference genome, allowing for isoforms of the same gene to be distinctly identified from different gene families,” the scientists write. Their analysis generated a highly diverse set of isoforms, which the authors believe “represents a nearly complete transcriptome of the hummingbird liver.”
With that dataset, the scientists found genes unique to hummingbird. “These genes showed a specific enrichment for pathways involved in lipid metabolism — suggesting that the hummingbird has evolved variants of these genes to achieve its high levels of metabolic efficiency,” they report.
The scientists note that follow-up functional assays will be an important next step in understanding and verifying the function of many genes of interest.