genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
Pathogen Gene Expression Profiling
Tiling Array Expression Profiling
Tiling array design
The “080604_SA_JH_Tiling” array was designed by Hanne Jarmer (Center for Biological
Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Lyngby,
Denmark) using algorithms, which have recently been published in the context of a B. subtilis
tiling array (Rasmussen et al. 2009). In brief, iso-thermal probes of 45 nucleotides (nt) to 65 nt
were designed to cover the whole genome of S. aureus. Probes were arranged in 22 nt intervals
on each strand and possessed an offset of 11 nt between the strands. The array design for
S. aureus was part of the international cooperation EU-IP-FP6-project BaSysBio (LSHG-CT2006-
037469) which is funded by the European Commission and coordinated by Philippe Noirot (INRA,
Mathématique Informatique et Génome, Jouy-en-Josas, France).
The sequences were synthesized on quartz wafers by NimbleGen (Roche NimbleGen,
Madison, WI, USA) in a custom, high-density DNA array format by applying the Maskless Array
Synthesizer (MAS) technology in combination with photo-mediated synthesis chemistry. The
basic principle of the synthesis is a solid-state array of miniature aluminum mirrors which direct
UV-light at the place where the next reaction steps should occur. An UV-labile protection group is
separated from the nascent oligonucleotide and releases a reactive site for binding of the next
nucleotide. Thus, the synthesis of the oligonucleotide sequences requires m x 4 reaction steps,
where m is the length of the oligonucleotide (number of nucleotides) and at each intermediate
length step the four possible nucleotides A, T, C, and G will be added in a single, separate
reaction.
Tiling array hybridization
The tiling array hybridization was performed at NimbleGen (Roche NimbleGen, Madison, WI,
USA) according to standard protocols. Briefly, 10 µg of high quality RNA samples were reverse
transcribed to cDNA in the presence of actinomycin D with subsequent alkaline RNA hydrolysis.
Precipitated and resolved cDNA was labeled with Cy3 dye via NHS-Ester Dye Coupling Reaction.
After spin-column based purification and quality control, labeled cDNA was hybridized together
with appropriate controls to tiling arrays of the 080604_SA_JH_Tiling design. Arrays were washed
and scanned and raw intensity data of tiling probes were provided to the customer.
Tiling array raw data analysis
The raw data analysis and condensing of probe data to transcripts/genes was performed by
Pierre Nicolas, Aurélie Leduc, and Philippe Bessières (INRA, Mathématique Informatique et
Génome, Jouy-en-Josas, France). Intensity values for annotated genes were derived from the
individual probe data by calculating the median of the probes located within the genomic
coordinates of these genes. Analysis of hybridization signal, identification of transcription start
and end boundaries and by this means segmentation into transcriptional units was executed by a
novel algorithm based on a hidden Markov model, which allows to identify new, formerly
unknown or non-annotated transcripts (Nicolas et al. 2009).
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