Computational protocol: Characterization and Differential Gene Expression between Two Phenotypic Phase Variants in Salmonella enterica Serovar Typhimurium

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Protocol publication

[…] Total DNA was isolated from S. Typhimurium strain 798 using the DNEasy Kit (Qiagen, Valencia, CA). The genome was sequenced to a depth of 30× coverage using pyrosequencing on Roche 454 with Titanium technology (Branford, CT). Sequence reads were assembled into contigs with Newbler Assembler and was finished and checked for consistency using PCR . The genome was annotated using the xBASE bacterial genome annotation service . Gene predictions were performed using Glimmer , tRNA genes were identified with tRNAScan-SE , and ribosomal RNA genes were identified with RNAmmer . Genomic comparisons were performed using MAUVE . Single nucleotide polymorphisms were identified using SNPsFinder . The completed sequence of strain 798 is deposited in Genbank under accession numbers CP003386 (chromosome) and CP003387 (plasmid). [...] Comparisons of rates of phase variation and invasion were analyzed for statistical significance in Excel (Microsoft, Inc., Redmond, WA) using the Student’s T test. The RNA-Seq data was analyzed using DEGseq (Bioconductor, Seattle, WA). Sequences were aligned using the published sequence for S. Typhimurium strain LT2 and the number of reads per gene was normalized as a percent of the total sequences obtained for that sample. The data was binned by gene and used as the measure of gene expression. Each strain had a biological duplicate. Criteria for significant differential gene expression was set at a p value of less than 0.05 and a minimum of a two-fold difference in expression. Comparisons were between i518 and i519 and i519 and SL1344. […]

Pipeline specifications

Software tools Newbler, Glimmer, tRNAscan-SE, RNAmmer, Mauve, DEGseq
Databases xBASE
Applications Genome annotation, RNA-seq analysis, Nucleotide sequence alignment
Organisms Salmonella enterica subsp. enterica serovar Typhimurium, Sus scrofa