Computational protocol: Unravelling the relationship between the tsetse fly and its obligate symbiont Wigglesworthia: transcriptomic and metabolomic landscapes reveal highly integrated physiological networks

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Protocol publication

[…] Bacteriomes were dissected from females at around 20 days post-eclosion. Three biological replicates of 10 bacteriomes were collected. Dual RNA-seq libraries were prepared with ScriptSeq Complete Gold Kit (Epidemiology) (Epicentre, Madison, WI) and sequenced (75 bp single-end read) on Illumina HiSeq 2000 by Yale University Center of Genome Analysis (YCGA, New Haven, CT). Reads were mapped to Wigglesworthia genome (NC_016893) and to Glossina transcripts (version 1.4 obtained from Vectorbase [], respectively, using CLC Genomics Workbench (CLC Bio, Cambridge, MA). We used the RPKM as a measure of relative gene expression []. Bacteriocyte-enriched Glossina genes were selected by comparing the ratio of RPKM expression values of transcripts between bacteriome-specific and previously published whole midgut transcriptomes []. Glossina transcripts were identified as enriched using the following parameters: bacteriome RPKM/whole gut RPKM ratio of greater than 5 and an average bacteriome RPKM of greater than 50. Statistical significance of gene enrichment was evaluated using the LOX software package []. Transcripts determined to be bacteriocyte enriched were annotated with GO terms using the Blast2GO software package []. Wigglesworthia-specific gene expression profiles were performed with KEGG pathway analysis []. Sequencing data are available in the Sequence Read Archive (SRR3956922-7) and detailed study protocols are described in the electronic supplementary material. […]

Pipeline specifications

Software tools CLC Genomics Workbench, Blast2GO
Databases SRA VectorBase Dual RNA-Seq KEGG PATHWAY
Application RNA-seq analysis
Organisms Drosophila melanogaster, Toxoplasma gondii, Wigglesworthia glossinidia
Chemicals S-Adenosylmethionine