1. Nature Genetics Vol.36 Sept 2004 Detection of Large-scale Variation In the Human Genome Iafrate, Feuk, Rivera, Listewnik, Donahoe, Qi, Scherer, Lee any image here Title Page Trevor Pugh MEDG505 – Genomic Analysis University of BC

2. Large-scale Copy-Number Variations: 5 W’s & How W hat are Large-scale Copy Number Variations? W ho have Large-scale Copy Number Variations? W here are Large-scale Copy Number Variations? W hy are there LCVs? W hen did LCVs arise? H ow are LCVs generated?

3. What are Large-scale Copy-Number Vars.? @ gains or losses of kb’s-Mb’s of genomic DNA @ seen in phenotypically normal individuals > may not be direct cause of genetic disease > may give rise to disease?  chromosomal rearrangements  subtle phenotypic variation (gene expression) @ NOT limited to intergenic or intronic regions

4. Who have Large-scale Copy-Number Vars? 1 1 1 1 1 1 1 1 1 1 2 1 1 1 Deletion! Five steps of CGH @Isolation of Genomic DNA from two patients/sources @Digestion of Genomic DNA into short fragments @Labeling of each sample with DIFFERENT detectable dyes @SIMULTANEOUS Hybridization of labeled probes to targets @Quantification of amount of hybridization to each target @ applied array CGH to 55 unrelated individuals @ spots are large DNA fragments ‘tiled’ ~1MB across the genome  low resolution, captures 12% of genome

5. @ applied array CGH to 55 unrelated individuals > 39 healthy phenotypes (normal karyotype) > 16 with known chromosomal abnormalities... against a pool of (different) normal controls @ detected *all* known LCVs in 16 abnormal samples > good sensitivity and specificity @ on average, detected 12.4 LCVs in normal controls! > mostly single large insert clones  up to 2Mb affected! Who have Large-scale Copy-Number Vars?

6. @ on average, detected 12.4 LCVs in normal controls # of LCVs found in: (False Positive rate <1 per 5,264 clones) @ 26 mapped to previously recognized segmental dupes @ 13 reside within 100kb gaps of current human genome sequence presentation  complicates assembly of final HGP sequence 102 Who have Large-scale Copy-Number Vars?

7. Everywhere! RED = copy gains BLUE = copy losses Where are Large-scale Copy-Number Vars?

8. @ across the genome involving as much as 2MB @ not limited to intergenic or intronic regions > can include duplicate genes! @ confined to localized chromosomal regions Where are Large-scale Copy-Number Vars.? OR > NOT duplication events of other chromosomes > tandem copy-number changes

9. @ tandem copy-number changes > visualized using FISH Where are Large-scale Copy-Number Vars.? RELATIVE: LossesNormalGains signals only from sister chromatids > CN gains show more head-to-tail gene copies RED = 5’ head GREEN = 3’ tail & fibre-FISH 6 duplicates9 duplicates12 duplicates

10. Where are Large-scale Copy-Number Vars.? @ tandem copy-number changes > verified by QT-PCR > ratios track FISH & CGH ratios almost perfectly!

11. Why do Large-scale Copy-Number Variations Exist? @ Unknown! When did Large-scale Copy-Number Variations Arise? “Large-Scale Variation Among Human and Great Ape Genomes Determined by Array Comparative Genomic Hybridization” Locke, Segraves, Carbone, Archidiacono, Albertson, Pinkel, Eichler Genome Research Vol 13, Issue 3, 347-357, March 2003 @ gene-rich regions susceptible to CN changes between humans and great apes @ local repatterning of hominoid chromosomes in euchromatic regions > duplication-driven mechanism of genome evolution @ Unknown!

12. How do LCVs Impact Us? @ new explanations for individual uniqueness > lineage studies? > forensics? @ increased gene copy number associated with increased susceptibility to diseases? > use as genetic markers? @ added level of complexity to the genome > comparative genomics? > phylogenetics? @ simplification of the genome > less regulatory networks, more ‘dosage effects’

13. Summary: Large-scale Copy-Number Variations (LCVs) W hat? @ gains or losses of kb’s-Mb’s of tandem genomic sequences W ho? @ varies from person to person, 12.4 LCVs on average W here? @ throughout the genome, localized to chromosomal regions @ not limited to intergenic or intronic regions W hy? @ Unknown! Conjecture: Gene evolution? Regulatory? W hen? @ Unknown! Conjecture: Complex organisms? Plant/Animal? H ow? @ new basis for human uniqueness & treatment @ new ways of interpreting the genome and its interactions

14. Questions? Questions @ Why do LCVs exist? When did they arise? @ Are these LCVs random? If not, what types of genes would have copy number variation? @ What possible mechanisms could create LCVs? @ Is this a genomic simplification or an added level of complexity? @ What are the implications of LCVs for current technologies, databases, and assemblies? > PCR, gene expression, genetics? > GenBank, OMIM? >Human Genome Project?

15. Extra Figures