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[…] The sequence of Ss-riok-1 was compared by BLASTx with sequences in non-redundant databases from NCBI (http://www.ncbi.nlm.nih.gov/) to confirm the identity of genes isolated. The translation of cDNA of Ss-riok-1 into predicted amino acid sequences was performed by free software Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html#downloads). The protein motifs of Ss-RIOK-1 were identified by scanning the databases PROSITE (www.expasy.ch/tools/scnpsit1.html) and Pfam (www.sanger.ac.uk/Software/Pfam/). Ss-RIOK-1 was aligned with the homologues from selected species using the program MAFFT 7.0 (http://mafft.cbrc.jp/alignment/software/), and the functional domains and subdomains were identified in the protein alignment. Promoter elements in the 5′-UTR were predicted using the transcription element search system Matrixcatch (http://www.gene-regulation.com/cgi-bin/mcatch/MatrixCatch.pl) .For phylogenetic analysis, the amino acid sequences of 27 homologues were retrieved from GenBank databases and the alignment of protein sequences was carried out by Clustal X and manually adjusted. The species selected were nine nematodes, including Ascaris suum (ERG87084.1), Brugia malayi (EDP30009.1), Caenorhabditis briggsae (CAP24959.2), Caenorhabditis elegans (CCD67367.1), Caenorhabditis remanei (XP_003098834.1), Haemonchus contortus (ADW23592.1), Loa loa (XP_003135673.1), Trichostrogylus vitrinus (CAR64255.1), Wuchereria bancrofti (EJW88234.1), and 14 non-nematode species, including Aedes aegypti (XP_001661999.1), Arabidopsis thaliana (NP_851100.1, AAM65700.1, NP_180071.1), Canis familiaris (XP_535878.1), Danio rerio (NP_998160.1), Drosophila melanogaster (NP_648489.1), Homo sapiens (EAW55210.1, NP_113668.2), Mus musculus (NP_077204.2), Oryza sativa (BAC79649.1), Pan troglodytes (XP_527225.2), Pongo abelii (CAH93232.1), Rattus norvegicus (NP_001093981.1, AAH79173.1), Saccharomyces cerevisiae (CAA99317.1), Xenopus laevis (NP_001116165.1), Xenopus tropicalis (XP_004915351.1). The phylogenetic analysis was conducted using the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods based on Jones-Taylor-Thornton (JTT) model in the MEGA v.5.0 . Confidence limits were assessed by bootstrapping using 1,000 pseudo-replicates for NJ, MP and ML, and other settings were obtained using the default values in MEGA v.5.0 . A 50% cut-off value was implemented for the consensus tree. [...] The S. stercoralis PV001 line, derived from a single female worm of the UPD strain , was maintained and cultured as described previously , , . S. stercoralis PV001 developmental stages were isolated using established methods , and included: free-living females (FL Female), post free-living first-stage larvae (PFL L1), infective third-stage larvae (iL3) (heterogonically developed), in vivo activated third-stage larvae (L3+), parasitic females (P Female), post-parasitic first-stage larvae (PP L1), and post-parasitic third-stage larvae (PP L3). Transcript abundances were quantified using RNAseq . Briefly, raw reads were aligned to S. stercoralis genomic contigs (6 December 2011 draft; ftp://ftp.sanger.ac.uk/pub/pathogens/HGI/) using the program TopHat2 v.2.0.9 (http://tophat.cbcb.umd.edu/) , employing the Bowtie2 aligner v.2.1.0 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) and SAMtools v.0.1.19 (http://samtools.sourceforge.net/). Transcript abundances were calculated using Cufflinks v.2.0.2 (http://cufflinks.cbcb.umd.edu/) as fragments per kilobase of coding exon per million fragments mapped (FPKM), with paired-end reads counted as single sampling events . FPKM values for coding sequences (CDS), ±95% confidence intervals, were calculated for each gene using Cuffdiff v.2.0.2. Significant differences in FPKM values between developmental stages and p-values were determined using Cuffdiff v.2.0.2, a program with the Cufflinks package , ; p-values <0.05 were considered statistically significant. […]

Pipeline specifications

Software tools BLASTX, BioEdit, MAFFT, MCatch, Clustal W, MEGA-V, TopHat, Bowtie2, SAMtools, Cufflinks
Databases Pfam PROSITE ExPASy
Applications Genome annotation, Phylogenetics, RNA-seq analysis, GWAS
Organisms Strongyloides stercoralis, Canis lupus familiaris, Saccharomyces cerevisiae, Homo sapiens
Diseases Nematode Infections