De novo sequence assembly and characterisation of a partial transcriptome for an evolutionarily distinct reptile, the tuatara (Sphenodon punctatus)

Abstract

Background: The tuatara (Sphenodon punctatus) is a species of extraordinary zoological interest, being the only surviving member of an entire order of reptiles which diverged early in amniote evolution. In addition to their unique phylogenetic placement, many aspects of tuatara biology, including temperature-dependent sex determination, cold adaptation and extreme longevity have the potential to inform studies of genome evolution and development. Despite increasing interest in the tuatara genome, genomic resources for the species are still very limited. We aimed to address this by assembling a transcriptome for tuatara from an early-stage embryo, which will provide a resource for genome annotation, molecular marker development and studies of development and adaptation in tuatara.

Results: We obtained 30 million paired-end 50 bp reads from an Illumina Genome Analyzer and assembled them with Velvet and Oases using a range of kmers. After removing redundancy and filtering out low quality transcripts, our transcriptome dataset contained 32911 transcripts, with an N50 of 675 and a mean length of 451 bp. Almost 50% (15965) of these transcripts could be annotated by comparison with the NCBI non-redundant (NR) protein database or the chicken, green anole and zebrafish UniGene sets. A scan of candidate genes and repetitive elements revealed genes involved in immune function, sex differentiation and temperature-sensitivity, as well as over 200 microsatellite markers.

Conclusions: This dataset represents a major increase in genomic resources for the tuatara, increasing the number of annotated gene sequences from just 60 to almost 16,000. This will facilitate future research in sex determination, genome evolution, local adaptation and population genetics of tuatara, as well as inform studies on amniote evolution.

Figure 1

Length distribution (A) and mean coverage per base (B) of assembled transcripts. White bars show data from all transcripts (32,911 sequences), while grey bars show data from annotated sequences only (15,965 sequences). The line denotes mean length (A) or coverage (B) for all transcripts (dotted line), and annotated sequences only (solid line).

Figure 2

Gene Ontology (GOslim) assignments for tuatara transcripts. Level 2 annotations are shown for the biological process and molecular function graphs, and level 7 annotations for the cellular component graph.