Methods cont –

1. Aim: To identify differentially expressed genes in different anatomical sites – identify genes targets required for in vivo adaptation • Intro – • Data exist to support the notion of differential gene expression in Staphylococcus aureus. • Many different genomes are available as references, that are well annotated – one will be chosen to reflect genotype of the colonizing (Nasal) and infecting (Bloodstream) isolates which will be isogenic – Reference genome gff file will be generated using Perl script. • For the aim of this experiment, transcript data will be simulated in the form of FastQ files – TA Help required here. • Methods – • Using simulated datasets, RNA sequences in FastQ format will be submitted to assembly pipeline which will use Perl scripts when possible and consist of : • - Tophat (Alternatively MapSplice, SpliceMap and RUM) will be used to align reads to reference genome (Reference genome will be indexed). • Readcount as a measure of transcript abundance will be estimated using Htseq and Cufflinks – Readcount will be used to call for differential expression by DESeq (or other method). This will identify differentially expressed genes (Top ~ 50 will be used for further analysis). • Visualization will be done using Integrated Genome Viewer (IGV) and Gbrowse. • Once reads are assembled into contigs, annotation will be achieved using Cufflinks to get gene identities and co-ordinates.

2. Methods cont – • - Time permitting further annotation will be done using databases (such as COG) to generate groups of related genes – will give an estimate of how gene families are differentially expressed in various sites. • Expected results - • Will be able to identify genes that are upregulated in different body sites and may reflect adaptation of the bacteria to different sites in the body. • Genes required for the invasion of the bloodstream may be identified. • Gene required for the colonization and persistence of the bacteria in the nasal cavity will be identified. • Submitted with happiness by the three musketeers – Julia, Davida and Frida • 10/25/2011 – 11.35 pm • After two gin and tonics and one glass of red-wine