1. Next Generation Microarray Technology: Overview of Febit’s Geniom One ® H. Bjørn Nielsen Center for Biological Sequence analysis Technical University of Denmark NORFA 2004 course Lo-skolen - May 21

2. Microarrays - The Technologies Stanford microarrays Affymetrix Febit Affymetrix Febit Stanford microarrays

3. aRNA Design Probes Import into Geniom One ® Over Night Synthesis Chip SAMPLE KONTROL FEBIT

4. The Technologies - Costs Facility setup cost: Stanford microarrays < 1 mill kr Affymetrix< 2 mill kr Febit< 2.5 mill kr Cost pr. transcript: Stanford microarrays < 0.5 kr Affymetrix< 0.5 kr Febit< 2.5 kr

5. The Technologies - Flexibility Stanford microarrays: Are flexible, but new probes must be ordered each time Affymetrix arrays: Are not flexible Febit arrays: Are highly flexible, the probes are synthesized over night inside Geniom One ® from an imported template file

6. The Technologies - Data Quality Reproducibility of data - the pearson correlation Stanford microarrays: 0.80-0.95 Affymetrix: > 0.95 Febit:> 0.95

7. The Technologies - Choice of Stanford microarrays: If you work with a limited number of species - maybe not yet sequenced - inexpensive - variable data quality Affymetrix: If you work with “standard” species - expensive - high quality of data Febit: If you work with many different species with available sequence information and/or wishes to perform iterative experiments - expensive - high quality of data

8. 8 arrays with 6,776 probes >54,000 probes pr. processor The Geniom One ® Processor

9. Photolithography - Micromirrors

10. TTT T T T T T T T A A A A A A A AAA Photolithography in situ synthesis Spacers bound to surface with photolabile protection groups Mask #1 Mask #2

11. Output from Geniom one

12. A Febit experiment - Goal and Idea Find all Transcription Start Sites (TSSs) in B. subtilis - We do this by the use of the Geniom One ® - A two-step method: - Dichotomy - Pin-point

13. 1. The Dichotomy Method 2. run: 3. run: 4. run: +1 1. run: 30-50 bp between probes covering all intergenic regions in the genome

14. The Problem of Tiling +1 Intensity Position

15. Castle et al., Genome Biol. 2003; 4 (10): R66 Genome studies, Bioinformatics, Methods, Genetics Probe-Intensity Profiles for Intron-Exon Boundaries Tiling of 35 mers

16. We Need Increased Specificity We know from physical chemistry that: DNA:DNA and RNA:DNA hybridizations are destabilized by mismatches or gaps, and that this is less pronounced for distal positions. Position Contribution to stability Surface end Middle Solution end

17. fragmentation Geniom ® one +1 2. The Pin-point Method

18. ATATAGGAGG TATAGGAGGA ATAGGAGGAT TAGGAGGATC CCAGGTAC...........TTCAATATAGGAGGATCATATGCCAG........ +1 3. The Pin-point Method Probes

19. The Hole Expressome? - What RNA should we use? - Analyzed existing microarray data - Found that most genes were represented in: - Heat shock experiment - 4 different time points in minimal medium

20. Transcription - What will we see? - How pronounced are the “holes” between the genes ??????

21. Labeling of the RNA - Two types of labeling: - 5’ label after fragmentation - 5’ label before fragmentation

22. Acknowledgements Hanne Ø. Jarmer Hanni Willenbrock Steen Knudsen Hans Henrik Saxild Kristine Bøje Dahlin