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Commentary

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Commentary

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  1. Commentary • Do not get too worried about "methods" and details. I fully expect there to be concepts and techniques that you simply are not going to know. • Try to figure out the "big picture." • Finding papers (from campus you will have access to a lot more journals) http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed http://www.google.com/scholar PubMed is better for finding more recent papers (ordered by date). Google/scholar is nice for finding papers that reference a paper When capturing figures from papers, blow up the figure as large as possible, capture, then shrink to fit slides (not the other way around). Spelling, grammar, syntax -- all count.

  2. Segmental Duplications and Copy-Number Variation in the Human Genome Sharp et al. Am. J. Hum. Genet. 77:78-88, 2005 presented by: Terry Braun

  3. Abstract • Human genome contains blocks of duplicated sequence (segmental duplications) • Identified 130 potential rearrangement hotspots (bioinformatically?) • Created a "microarray" to assess copy-number variations • Examined 47 "normal" individuals • Identified 119 regions of copy-number polymorphism (CNP)

  4. Outline • Background • CGH • Segmental duplications • Materials and Methods • very brief • Results • CNPs in the population • Validation • Discussion

  5. Background • Segmental duplications • blocks of DNA -- 1kb to 400 kb • 5% of genome is duplicated • Implicated in more than 25 genomic disorders • Array comparative genomic hybridization (CGH)

  6. Background (cont) • Referenced -- Representational Oligonucleotide Microarray Analysis (ROMA) • Nice description on wikipedia • Main difference -- uses restriction enzymes on the test and reference genome -- which improves efficiency

  7. Background (cont) • Previous work had limited coverage of whole genome. • Hypothesis: regions flanked by segmental duplications are susceptible to rearrangement -- "hotspots" of genome instability that results in "copy-number variations"

  8. Background (cont) • Utilized knowledge from previous studies on genomic disorders and copy number variations: • regions flanked by duplications • duplications > 10K • >95% sequence identity • region spanned is 50kb to 10Mb • Searched genome for regions with these characteristics (in silico) • Disappointed in lack of details

  9. Figure 1

  10. Material and Methods • 130 regions found • Selected "BACs" • Bacterial artificial chromosomes • Technique for keeping and duplicating portions of the genome • Used by human genome project • 100kpb - 350 kbp • Analysis used to select BACs

  11. BAC Selection Priority • BACs contained in "hotspot" • BACs overlapping segmental duplications • flanking BACs in peripheral unique sequence as local control Selected 2,194 BACs (192 single copy, randomly selected)

  12. Microarry Comparative Genome Hybridization • glass slide (or silicon waffer) • DNA is spotted (or grown) • High density (1000's to millions of "spots" or probes) • Millions of copies per "spot"

  13. Source: www.bioteach.ubc.ca/MolecularBiology/microarray/ graphics by Jiang Long

  14. Results • Validation • true positive • strong correlation to BAC's that "lit up" in 3 individuals with known CNV's, and proportional to number of copies • false positive • 4 self-vs-self (assuming they used the BACs???) • threshold of log2 hyb ratios deviating > 2 standard deviations from mean correspond to FPR ~3 per 4000

  15. Results • 160 variant BACs in 47 individuals (corresponding to 119 nonredundant regions)

  16. Copy Number Polymorphisms (CNP)

  17. More Validation -- Individual

  18. FISH Validation – Segmental Duplication BAC

  19. Validation – Chrm 8 BACs

  20. Analysis of CNPs • 15.7% of "hotspot" regions were variant • 4.1% non-hotspot regions were variant • => 3.8-fold enrichment • Chi^2 = 73.8 • P < 0.000001 • CNV regions were "not particularly gene poor."

  21. Discussion • "4-5-fold enrichment of CNPs flanked by SD's • Supports notion that regions are predisposed to rearrangement • Multi-ethnicity speculations • evolutionarily ancient, prior to separation of groups, OR • recurrent events that occurred independently in multiple founders

  22. Supplemental Data

  23. Conclusions • Duplications may be impediment to genome assembly • UCSC genome view of CNP • http://www.som.soton.ac.uk/research/geneticsdiv/anomaly%20register/ • Hotspots represent excellent candidate sites of recurrent rearrangements associated with novel genomic disorders

  24. Summary • Comparative genome hybridization • 47 individuals • Copy number polymorphisms found • Enriched for CNPs by examining segmental duplications (a bioinformatics approach) • Appears to be convincing • Resolution leaves a lot to be desired • Difference between “hotspot” and “segmental duplications” regions not clear