Assembling the Transcriptome of the Toadlet Brachycephalus garbeana (Amphibia: Anura: Brachycephalidae)

The species of Brachycephalus display diversification of several morphological features, such as skin color, body size miniaturization and ossification patterns. They are endemic to the Atlantic Forest of Brazil and most of them occur in allopatry. This biological scenario raises intriguing questions about the biogeography and the evolution of Brachycephalus, which could partially be addressed through deep sequencing approaches using next-generation methods and transcriptome assembly to define genes and molecular markers. This information should be useful for phylogeny, adaptation, expression and population structure studies. Here we report the first analyses towards the characterization of a Brachycephalus transcriptome.

RNA (intestine and stomach) from a Brachycephalus garbeana female was used for Illumina paired-end sequencing, which generated over 180x10⁶ reads. By means of the CLC Genomics Workbench, nine de novo assemblies with varying k-mers (24 to 55) were conducted using a minimum contig length of 450 bp. The contig number varied from ~14,500 to ~38,000. N50 values varied from 948 to 1,293bp. In order to evaluate the assemblies, a blastx analysis using the protein data of Xenopus tropicalis and X. laevis was performed. The blast outputs were analyzed with the MuSeqBox software and revealed a mean percentage of hits of 55%, a mean query length of 1,194bp and query coverage ranging from 69 to 76% (top 1 HSPs only).  Approximately 3% of all contigs contains putative full coding sequences. To further verify the quality of our assemblies, we intend to apply alternative algorithms to our data prior to proceeding to gene annotation.