Norman Pavelka (Rong Li lab)

1. Technology & Methods Seminar: “Tiling Arrays - Probing Genome and Transcriptome Structure” On the use of Affymetrix Tiling Arrays for Comparative Genomic Hybridizations Norman Pavelka (Rong Li lab) March 29, 2007

2. Background: Role of MYO1 in cytokinesis Myo1 • Phenotype of yeast cells experiencing an acute loss of MYO1: • Severe cytokinesis defect • Impaired cell viability • Phenotype of yeast cells experiencing a chronic loss of MYO1: • Extremely heterogenous • Occasionally: full recovery of cytokinesis proficiency and of growth ability

3. Biological question: What genome changes occurred in e-strains? • Polyploidization? • Aneuploidization? • Interstitial deletions? • Reciprocal translocations? • Non-reciprocal translocations? • Amplifications? • Single-nucleotide mutations? Albertson & Pinkel, Hum Mol Genet (2003)

4. Method: array-based Comparative Genomic Hybridization (aCGH) U.C. Berkeley Division of Biostatistics Working Paper Series (2002), paper 106.

5. Technology: Affymetrix Yeast Tiling Arrays ~6.5 million unique probes on the chip Gresham et al., Science (2006) ~12.5 million bp in the yeast genome Designed to interrogate the yeast genome with a 5bp resolution:

6. Experimental protocol: Strain 2b (wt) Ladder 75 mU DNase I 150 mU DNase I 25 500 2000 6000 Fragment length (nt) 200 1000 4000 Strain 7a-1 • Extraction of genomic DNA with Phenol / Chloroform / Isoamylalcohol • “Controlled” fragmentation with DNase I (5 min at 37 °C) • End-labeling with TdT and biotin-dUTP • Hybridize on Affy chips • Stain with streptavidin-PE • Wash and scan chips

7. 2b (low DNase I, large fragments) 7a-1 (low DNase I, large fragments) 2b (high DNase I, small fragments) 7a-1 (high DNase I, small fragments)

8. Limitations: What genome changes can we see by aCGH? • Polyploidization? • Aneuploidization? • Interstitial deletions? • Reciprocal translocations? • Non-reciprocal translocations? • Amplifications? • Single-nucleotide mutations? Albertson & Pinkel, Hum Mol Genet (2003)

9. Observation #1: Deletion of the MYO1 locus MYO1 locus +1 0 log2(ratio) -10 +1 0 log2(ratio) -10 Chromosome VIII

10. Observation #2: “Duplication” of the TRP1 locus TRP1 +10 log2(ratio) 0 -1 +10 log2(ratio) 0 -1 Chromosome IV Caveat #1: No information on where the signal comes from!

11. Caveat #2: Highly repetitive sequences! (aka “Saturation” effect) +1 log2(ratio) 0 -1 +1 log2(ratio) 0 Full-length Ty1 Full-length Ty1 Ty1 LTR -1 Chromosome II

12. Observation #3: Gradual loss of signal towards telomeres +1 log2(ratio) 0 -1 +1 log2(ratio) 0 -1 Full sequence of chromosome II

13. Observation #4: Aneuploidies Chr.

14. Caveat #3: “Dilution” effect

15. Possible observation #1: Non-reciprocal translocations? Dunham et al., PNAS (2002)

16. Possible observation #2: Single-nucleotide changes? +10 log2(ratio) 0 -10 35 45 0 5 10 15 20 30 40 25 Probes on the chip Genomic DNA 35 45 0 5 10 15 20 30 40 25 Gresham et al., Science (2006)

17. Summary: • What can be seen by CGH on Tiling Arrays? • Anything that causes a change in the copy number of a DNA segment, e.g. aneuploidies, deletions/amplifications, non-reciprocal translocations, etc. • Mutations that affect the hybridization of multiple overlapping probes, i.e. single-nucleotide changes. • What can not be seen by CGH? • Anything that does not cause a change in the copy number of a DNA segment, e.g. polyploidization, reciprocal translocations etc. • If probes are too long and non-overlapping, single-nucleotide mutations will not be detectable. • What are the most common pitfalls? • No information about where the signal actually comes from! • No reliable information from probes hybridizing to highly-repetitive sequence (because of “saturation” effect)! • If some chromosomes are gained or lost, this will affect the log-ratios also of all other chromosomes (because of “dilution” effect)!

18. Acknowledgements: • Microarray group: • Karin Zueckert-Gaudenz • Allison Peak • Chris Seidel • Rong Li lab: • Giulia Rancati • Rong Li