Full-length, Single Molecule, Whole Transcriptome cDNA Sequencing of the European Cuttlefish

Alternative splicing dramatically increases the protein-coding potential of most eukaryotic organisms.  While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants.  The Iso-Seq™ protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for gene discovery and annotation.  As most transcripts range from 1 – 10 kb, fully intact RNA molecules can be sequenced using SMRT® Sequencing (avg. readlength:  10-15 kb) without requiring fragmentation or post-sequencing assembly.

We have employed the Iso-Seq method to generate a full-length, whole-transcriptome study for the European cuttlefish (Sepia officinalis). Cuttlefish are intelligent invertebrates, exhibiting extraordinary ability to change color and texture for camouflage or communication. They also regenerate limbs after injury, but the molecular bases for these functions are largely unknown.  Full-length cDNA molecules were sequenced from four cuttlefish tissues (brain, skin, arm, stellate ganglion).  cDNA libraries were size fractionated with BluePippin and converted into SMRTbell libraries.  Using a preliminary cuttlefish draft genome assembly, 37% of transcript reads mapped to the genome at >50% length, 22% at >90% length while 93% map at some length.  This study shows long reads from PacBio sequencing are well-suited for characterizing full-length cDNA spanning entire transcripts. This is particularly valuable where, as with cuttlefish, the genome assembly is fragmented due to polymorphism, overall size and repeat structure, and prior information about transcripts from this or any closely related species is unavailable.