Total RNA was mRNA purified using OligoTex mRNA extraction beads (Qiaqen) with the resulting purified mRNA being eluted in 40 μl of nuclease free water. All RNA samples were quality checked by gel electrophoresis on a 1.2% TAE agarose gel and by spectrophotometry using a Nanodrop spectrophotometer (LabTech International). Purified mRNA samples from regenerating check details arms of O. victoriae were pooled, in equal masses, for 454 sequencing on ¼ of a picotitre plate using the GS-FLX platform (Roche, Maryland, USA) at the DNA Sequencing Facility, Department of Biochemistry, University of Cambridge. The resulting sequence

reads were imported into Geneious (Drummond et al., 2010) for quality trimming and assembly into contiguous sequences (contigs). After quality trimming to a phred quality score equivalent of 20 (1% error chance per base) the remaining sequences were assembled using the assembler included in the Geneious software using the medium–low sensitivity option. Assembled contiguous sequences and singletons > 300 bases in length were imported into the Blast2GO program (Conesa et al., 2005) and compared to the NCBI non-redundant (nr) database using BLASTX with an E-value cut-off value of 1.0 e- 6 to identify transcripts with sequence similarity to known genes. These transcripts were further annotated using Gene Ontology (GO). Mapping of assembled sequence reads to known pathways and pathway map generation

was carried out using the KEGG Automatic Annotation Server (KAAS) with a minimum blast bit score of 60 for each alignment (Moriya et al., 2007). A phylogenetic tree to denote the Selleckchem LEE011 grouping of the putative Sox transcripts with known

Sox genes was carried out in Geneious (Drummond et al., 2010) using the Geneious tree builder plugin (Jukes-Cantor genetic distance Coproporphyrinogen III oxidase model , Neighbour-Joining tree building method without an outgroup). All sequence data were submitted to the NCBI SRA (short read archive) with the accession number: SRP013357.1 Assembly of the 454 pyrosequencing reads produced from the mRNA of regenerating arms of O. victoriae produced 18,003 contigs with an average size of 606 bp. There were also 31,947 singletons with an average size of 303 bp, of which 17,015 were > 300 bp in length ( Table 1), however, these were not included in the rest of this study. Of the 18,003 assembled contigs 3340 (19%) showed a blast match against the NCBI non-redundant database with an expected value cut off of 1.0 e− 6 ( Supplemental file 1). The low level of putative annotation was similar to that of pyrosequencing studies in other non-model invertebrate marine species ( Meyer et al., 2009, Clark et al., 2010, Clark et al., 2011 and Craft et al., 2010). In the blast search results 1240 of the 3430 matches (36% of the total) were to transcripts from the purple sea urchin Strongylocentrotus purpuratus ( Supplemental file 1).