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Tiling Arrays

Tiling Arrays. Technology and Methods Seminar March 29, 2007 . Madelaine Gogol Programmer Analyst Microarray mcm@stowers-institute.org. Norman Pavelka Postdoc Rong Li Lab nxp@stowers-institute.org. http://wiki/research/MadelaineGogol. Tiling Arrays - Overview. What is a tiling array?

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Tiling Arrays

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  1. Tiling Arrays Technology and Methods Seminar March 29, 2007 Madelaine Gogol Programmer Analyst Microarray mcm@stowers-institute.org Norman Pavelka Postdoc Rong Li Lab nxp@stowers-institute.org http://wiki/research/MadelaineGogol

  2. Tiling Arrays - Overview • What is a tiling array? • What can I do with it? • ChIP-chip • CGH • Expression • Which tiling arrays are available for my experiments? • in-house yeast tiling array (design details) • Agilent • Affymetrix • How will we analyze and visualize the data?

  3. What is a Tiling Array? Probe 1 Gene 1 A microarray with many probes distributed in an evenly spaced way across an entire genome.

  4. What can I do with tiling arrays? • Map the transcriptome • what’s being expressed? • ChIP-chip • where are proteins binding? • CGH • what are the differences in genome structure? • Other possibilities • Map the methylome • Genome resequencing • Polymorphism discovery

  5. http://www.ebi.ac.uk/huber-srv/queryGene/

  6. ChIP-chip Hybridize to Microarray PCR w/aa-dNTP Analyze image, calculate ratios

  7. array CGH Hybridize to Microarray

  8. In-house yeast tiling array (YOGie) • Covers the yeast genome • Just printed • resolution ~ 250 bases • freely available

  9. Spotted Microarray Manufacturing

  10. Operon Probe Set • 6307 probes, length 70 • Designed one per ORF, near 3’ end. • YOG arrays (yeast oligonucleotide) • YBOX: 3072 new probes

  11. Intergenic Probe Set: Design • Design target: yeast Intergenic regions • original goal: leave no area > 500 uncovered Gene 1 Gene 2 Fasta format 140-mer sequences tiling the intergenic regions Array Oligo Selector (AOS) 9,405 70-mer sequences from the intergenic regions

  12. Intergenic Probe Set • 9405 70-mer probes • No region greater than 360 left uncovered • ~ 220 bases between probes on average • Chromosome 3 completely tiled • YOGi arrays (YOG+intergenic)

  13. 5’ probe set: Design • Goal: • fill in gaps left by operon set in 5’ region of gene • leave no region > 500 without a probe • Targets: ORF regions 5’ of operon probe Array Oligo Selector (AOS) operon probe 5’ 3’ Gene 1

  14. 5’ probe set: Reduction • Too many probes • reduce to 6666 or less (budget and printing constraints) • Probes within 260 bases of eachother • winnowed • Tm • binding energy • number of matches to genome operon probe 5’ 3’ Gene 1

  15. 5’ probe set • 6,512 70-mer probes • tiles the region of each gene between the operon probe and the 5' end. • YOGie arrays (YOG+intergenic+enhanced)

  16. In-house yeast tiling array: YOGie Together, the operon, intergenic, and 5’ sets make up our homemade yeast tiling array • Freely available • Also includes tight tiles of • all centromeres • 7 sub-genomic regions 10-20 kb

  17. Agilent Microarray Manufacturing

  18. Agilent Tiling Arrays ChIP-on-chip Arabidopsis Whole Genome C. elegans Whole Genome Drosophila Whole Genome Human CpG Island Human ENCODE 244K Human Promoter Mouse Promoter Yeast Whole Genome 4 x 44K Yeast Whole Genome 244K Zebrafish Expanded Promoter Zebrafish Proximal Promoter Custom ChIP-on-chip Oligo aCGH Human Genome 244K Human Genome 105K Human Genome 44K Mouse Genome 244K Mouse Genome 105K Mouse Genome 44K

  19. Agilent Tiling Arrays: formats and cost per slide per hyb 244k 1 $400 $400 105k 2 $640 $320 44k 4 $720 $180 15k 8 $800 $100

  20. Agilent Custom Array Design • Take an agilent microarray design • Remove some probes • Put in your own probes • Design using agilent’s web application, earray. • You can also design everything from scratch

  21. earray http://earray.chem.agilent.com/

  22. Affymetrix Microarray Manufacturing

  23. Affymetrix Tiling Arrays Arabidopsis Tiling 1.0R ArrayC.elegans Tiling 1.0R ArrayChromosome 21/22 1.0 Array SetChromosome 21/22 2.0R ArrayDrosophila Tiling 1.0R ArrayENCODE01 1.0 ArrayHuman Genome Arrays +Mouse Genome Arrays +S. cerevisiae Tiling 1.0R ArrayS. pombe Tiling 1.0FR Array Cost ~ 500$ per array, so 500$ per hyb. http://www.affymetrix.com/support/technical/byproduct.affx?cat=exparrays

  24. Summary of Tiling Arrays (only yeast shown)

  25. Tiling array data analysis: still an adventure Affy TAS (Tiling Analysis Software) Agilent ChIP Analytics & CGH Analytics Other genome browsers, R packages, other people’s software, Do-it-yourself, perl, statistical models

  26. Data Visualization: Genome Browsers: UCSC http://wiki/research/Bioinformatics/GenomeBrowsers http://genome.ucsc.edu/cgi-bin/hgTracks?db=sacCer1&hgt.customText=http://research.stowers-institute.org/mcm/YOGie1.bed

  27. Data Visualization: Genome Browsers: UCSC

  28. Data Visualization: Genome Browsers: IGB http://wiki/research/IntegratedGenomeBrowser

  29. Data Analysis: Sliding windows ChIPOTle, PeakFinder, R scripts, etc.

  30. Data Analysis: Annotating and comparing peaks http://main.g2.bx.psu.edu/

  31. Data Analysis: Average Gene analysis Profile of binding across an average gene http://wiki/research/Microarray/AverageGeneAnalysis

  32. Summary • Tiling arrays • CHip-chip, CGH, expression • Which ones are available • In-house, Agilent, Affy • Data analysis • Future...

  33. The Future of Tiling Arrays The resolution continues to increase...

  34. Other Future Genomic Technology 454 and Solexa/Illumina “Next Generation” sequencing • “Sequence everything in the tube” • Shares some things with tiling arrays • even more unbiased • vast quantities of data • analysis methods are being developed

  35. Thanks!Microarray Bioinformatics All the labs that use microarrays!Bing LiWorkman lab Jennifer BuppJasperson lab Norman PavelkaRong Li Lab 5th floorBuilding 2 Bioinformatics Microarray N Allison Karin Me Brian Chris

  36. Technology & Methods Seminar “Adventures in Electron Microscopy” Rhonda Allen Histology Thursday, April 26th, 1:00 p.m. Classroom (1st floor, Administration Building) Schedule with abstracts and previous presentation slides can be found on: K:\Weekly Seminar Schedule\Thursday -- Technology & Methods Information regarding previous seminars can be found at: http://research.stowers-institute.org/wiw/external/Seminars/index.htm

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