The functional annotations based on primary sequence level was done using Interproscan (https://www.ebi.ac.uk/ interpro/), and further classified into different Gene Ontologies (GO). We further added various sub-categories within each GO-ontology and weighted them with FPKM (Fragments Per Kilobase of transcript per Million mapped reads, a measure of the relative expression of an individual transcript) values for each gene. Those transcripts highly expressed in the worm’s intestine were determined through comparison with data from the whole adult male transcripts available from the SRA for A. ceylanicum (accession numbers: SRX1116908 and SRX1116915) downloaded and employed in this study. The Merops database (https://merops.sanger.ac.uk/), a manually curated peptidase and inhibitor database, was used to annotate and classify peptidases in order to identify which proteases or peptidases are highly expressed in the hookworm intestine. Pepunit.lib sequences from release 9.12 were downloaded from the website (http://merops.sanger.ac.uk/download/) and used to create a custom Blastp database using BLAST command line tools and an e-value of less than 10-6. The hits were weighted by using their FPKM values and then summed to plot using proportional values. Molecules involved in transmembrane transport were identified using the TrSSP dataset (http://bioinfo.noble.org/ TrSSP/?dowhat=Datasets). The transporter sequences were downloaded from the Transporter Substrate Specificity Prediction Server (TrSSP) and used for BlastP searches against the hookworm intestinal RNA-Seq genes obtained in this study by setting an e-value of less than 10-6. Phylogenetic trees were generated for those functional protein homologues identified in this study using Phylogeny.fr [29] (http://www.phylogeny.fr/index.cgi). Signal peptides were predicted using Phobius Server (http://phobius.sbc.su.se/) [30]. The transmembrane domains were predicted using
Wei et al. Parasites & Vectors (2016) 9:518 Page 3 of 14
TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) [31].