European Cuttlefish Whole Transcriptome Sequencing: A Single-Molecule Full Length Transcript Survey with Iso-Seq Method

Cuttlefish are a part of several global cuisines.  They 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 all these functions are largely unknown.  We present a whole-transcriptome study for the European cuttlefish (Sepia officinalis), using Pacific Biosciences’s Iso-Seq method and RSII sequencing platform to generate long (>8kb) reads and full-length transcripts.  Assembly of full-length transcripts for novel genomes is challenging when using only short-read sequencing methods, especially when no closely related genome sequence is available to assist with annotation.   We sequenced full-length cDNA molecules from four cuttlefish tissues (brain, skin, arm, stellate ganglion) using Iso-Seq methods.  cDNA libraries were size fractioned with BluePippin. Each size fraction was converted into a separate SMRTbell library. Reads containing both 5’ and 3’ adapters were identified as full-length transcripts using IsoSeq analysis. On average, 72,000 reads were generated producing 17,500 full-length transcripts per SMRT cell.  Full-length transcripts are 1.0-6.4kb in length. Using a preliminary cuttlefish draft genome assembly, 37% of transcript reads mapped to the genome at >50% length, 22% at >90% length, and 93% at any length.  This study shows long reads from RSII 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.  We are using this approach to annotate various reference genomes.