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Open Access Highly Accessed Research article

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

Hilary C Miller1*, Patrick J Biggs2, Claudia Voelckel3 and Nicola J Nelson1

Author affiliations

1 Allan Wilson Centre for Molecular Ecology and Evolution, School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, 6140, New Zealand

2 Infectious Disease Research Centre (IDReC), Institute of Veterinary, Animal & Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand

3 Allan Wilson Centre for Molecular Ecology and Evolution, Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand

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Citation and License

BMC Genomics 2012, 13:439  doi:10.1186/1471-2164-13-439

Published: 31 August 2012

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.

Keywords:
Illumina sequencing; Transcriptome; De novo assembly; Sex determination; Repetitive elements; Tuatara; Sphenodon punctatus; Reptile genomics