Clustering was done with tclust, which proceeds by a transitive approach (minimum overlap: 60 bp at 20 bp maximum of the end of the sequence). Assembly was done with CAP3 (minimum similarity 94%). To detect unigene similarities with other species, several blasts (with high cut-off e-values) were performed against the following
databases: NCBI nr (blastx (release: 1 March 2011); e-value < 5, HSP length > 33aa), Refseq genomic database (blastn, e-value < 10), Unigene division Arthropods (tblastx, #8 Aedes aegypti, #37 Anopheles gambiae, #3 Apis mellifera, #3 Bombyx mori, #53 Drosophila melanogaster, #9 Tribolium castaneum; e-value < 5), Nasonia vitripennis Nvit OGS_v1.0 (CDS predicted by Gnomon (NCBI)) and Wolbachia sequences from Genbank (blastn (release 164); e-value < e-20). Gene Ontology annotation was carried out using Blast2go software [38]. During the first step (mapping), AZD5363 price a pool of candidate GO terms was obtained for each unigene by retrieving GO terms associated with the hits obtained after a blastx search against NCBI nr. During the second step (annotation), reliable GO terms were selected from the pool of candidate GO terms by applying the Score Function (SF) of Blast2go with permissive annotation parameters (EC_weight=1, e-value_filter=0.1, GO_weight=5, HSP/hit coverage cut-off=0%). In the third step of the annotation procedure, the pool of GO terms selected during the annotation step was
merged with GO terms associated with Interpro domain (Interpro predictions based on the longest ORF). Finally, Tofacitinib research buy the Annex augmentation step was run to modulate the annotation by adding GO terms derived from implicit relationships between GO terms [39]. In order to extract the
biological processes and molecular functions statistically over-represented in aposymbiotic libraries, we performed a hyper-geometrical test between GO terms from the aposymbiotic libraries (OA1 and OA2) and those from the OS library, which corresponds to natural physiological conditions. The p-values were then adjusted using Bonferroni’s correction. Pazopanib manufacturer To perform a functional enrichment analysis of the unigenes extracted from the SSH, we used the FatiGO web tool [40] on the OS library. With respect to the GO analysis, levels 3 and 6 were chosen to describe biological processes, and level 4 was chosen to describe molecular functions. Gene expression measurement by quantitative RT-PCR (qRT-PCR) We sought to determine the effect of symbiosis on the expression of a set of candidate genes involved in immunity, programmed cell death and oogenesis. For that purpose, we first compared gene expression between symbiotic and aposymbiotic samples, in ovaries (to characterize the dependence phenotype induced by Wolbachia) and then in males (to provide additional information concerning the specificity of the process). In order to limit the influence of the presence of eggs in symbiotic vs.