Computational protocol: Bifidobacterium thermophilum RBL67 impacts on growth and virulence gene expression of Salmonella enterica subsp. enterica serovar Typhimurium

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Protocol publication

[…] Illumina raw data reads (100 bp) were separated by barcode and mapped against the genome of RBL67 (GenBank accession no. CP004346) or Salmonella Typhimurium LT2 (GenBank accession no. AE006468) using the CLC Genomics Workbench 6.5.1 (CLCbio, Aarhus, Denmark) applying the default settings. Maximum allowance of mismatches was set at 2, minimum length fraction at 0.9 and minimum similarity fraction at 0.8.Statistical analysis for differential gene expression of the mono- and co-cultures was done with the statistical software R (http://www.R-project.org) using the GLM method [] included in the Bioconductor EdgeR software package [–] based on negative binomial distribution. Genes with low read numbers (sum of reads in all samples <3 counts per million (cpm)) or with high read numbers (number of reads >50,000 cpm in each sample) were filtered out before data normalization. A false discovery rate (FDR) value <0.05 and a differential expression of at least 2 fold (1 < log2 ratio < −1) was used as cut off for significant differentially expressed genes in mono-culture and co-culture []. Proteins of RBL67 and LT2 were assigned to gene ontology categories (GO) using Blast2GO at standard settings []. GO categories enrichment analyses were performed and visualized using the BiNGO plugin [] in Cytoscape (v.3.0.1, []) applying the hypergeometric test with Benjamini and Hochberg false discovery rate correction option. The significance cutoff for overrepresented gene ontology categories was a corrected p-value of <0.05.Virulence factors of Salmonella LT2 were identified by genome wide blast against the virulence factor database (VFDB) [], using a cut off E-value of 1−20. Significant enrichment of virulence factors was calculated using the Fisher’s Exact Test Calculator for 2 × 2 Contingency at www.research.microsoft.com/en-us/um/redmond/projects/mscompbio/fisherexacttest/.The RNAseq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [] and are accessible through GEO Series accession number GSE65716 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65716). […]

Pipeline specifications

Software tools CLC Genomics Workbench, CLC Assembly Cell, edgeR, Blast2GO, BiNGO
Databases GEO VFDB
Application RNA-seq analysis
Organisms Salmonella enterica subsp. enterica serovar Typhimurium, Bifidobacterium thermophilum, Homo sapiens
Diseases Salmonella Infections
Chemicals Amino Acids