Tiling Arrays

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Tiling Arrays
Technology and Methods Seminar March 29, 2007
Norman Pavelka Postdoc Rong Li Lab nxp_at_stowers-ins
titute.org

• Madelaine Gogol
• Programmer Analyst
• Microarray
• mcm_at_stowers-institute.org

http//wiki/research/MadelaineGogol
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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
• whats 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

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http//www.ebi.ac.uk/huber-srv/queryGene/
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ChIP-chip
Hybridize to Microarray
PCR w/aa-dNTP
Analyze image, calculate ratios
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array CGH
Hybridize to Microarray
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In-house yeast tiling array (YOGie)

• Covers the yeast genome
• Just printed
• resolution 250 bases
• freely available

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Spotted Microarray Manufacturing
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Operon Probe Set

• 6307 probes, length 70
• Designed one per ORF, near 3 end.
• YOG arrays (yeast oligonucleotide)
• YBOX 3072 new probes

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Intergenic Probe Set Design

• Design target yeast Intergenic regions
• original goal leave no area gt 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
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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 (YOGintergenic)

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5 probe set Design

• Goal
• fill in gaps left by operon set in 5 region of
  gene
• leave no region gt 500 without a probe
• Targets ORF regions 5 of operon probe

Array Oligo Selector (AOS)
operon probe
3
5
Gene 1
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5 probe set target selection

• What size targets?
• 355mers that overlap by 70

500
(500-70)/2 215
7021570355
70
target 1
target 2
215
215
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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
3
5
Gene 1
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5 probe set

• 6,512 70-mer probes
• tiles the region of each gene between the operon
  probe and the 5' end.
• YOGie arrays (YOGintergenicenhanced)

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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

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Agilent Microarray Manufacturing
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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
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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
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Agilent Custom Array Design

• Take an agilent microarray design
• Remove some probes
• Put in your own probes
• Design using agilents web application, earray.
• You can also design everything from scratch

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earray
http//earray.chem.agilent.com/
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Affymetrix Microarray Manufacturing
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Affymetrix Tiling Arrays
Arabidopsis Tiling 1.0R Array C.elegans Tiling
1.0R Array Chromosome 21/22 1.0 Array Set
Chromosome 21/22 2.0R Array Drosophila Tiling
1.0R Array ENCODE01 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/byprod
uct.affx?catexparrays
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Summary of Tiling Arrays (only yeast shown)
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(No Transcript)
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Tiling array data analysis still an adventure

• Affy
• TAS (Tiling Analysis Software)
• Agilent
• ChIP Analytics CGH Analytics
• Other
• genome browsers, R packages, other peoples
  software, Do-it-yourself, perl, statistical models

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Data Visualization Genome Browsers UCSC
http//wiki/research/Bioinformatics/GenomeBrowsers
http//genome.ucsc.edu/cgi-bin/hgTracks?dbsacCer1
hgt.customTexthttp//research.stowers-institute.
org/mcm/YOGie1.bed
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Data Visualization Genome Browsers UCSC
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Data Visualization Genome Browsers IGB
http//wiki/research/IntegratedGenomeBrowser
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Data Analysis Sliding windows
ChIPOTle, PeakFinder, R scripts, etc.
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Data Analysis Annotating and comparing peaks
http//main.g2.bx.psu.edu/
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Data Analysis Average Gene analysis

• Profile of binding across an average gene

http//wiki/research/Microarray/AverageGeneAnalysi
s
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Summary

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

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The Future of Tiling Arrays

• The resolution continues to increase...

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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

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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
Brian
Chris
Me
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Technology Methods Seminar

• Adventures in Electron Microscopy
• Rhonda Allen
• Histology
• Thursday, April 26th, 100 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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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
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Background Role of MYO1 in cytokinesis

• 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

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Biological question What genome changes occurred
in e-strains?
Albertson Pinkel, Hum Mol Genet (2003)
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Method array-based Comparative Genomic
Hybridization (aCGH)
U.C. Berkeley Division of Biostatistics Working
Paper Series (2002), paper 106.
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Technology Affymetrix Yeast Tiling Arrays
12.5 million bp in the yeast genome
Designed to interrogate the yeast genome with a
5bp resolution
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Experimental protocol

• 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

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2b (low DNase I, large fragments)
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Limitations What genome changes can we see by
aCGH?
Albertson Pinkel, Hum Mol Genet (2003)
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Observation 1 Deletion of the MYO1 locus
1
0
log2(ratio)
-10
1
0
log2(ratio)
-10
Chromosome VIII
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Observation 2 Duplication of the TRP1 locus
10
log2(ratio)
0
-1
10
log2(ratio)
0
-1
Chromosome IV
Caveat 1 No information on where the signal
comes from!
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Caveat 2 Highly repetitive sequences!
(aka Saturation effect)
1
log2(ratio)
0
-1
1
log2(ratio)
0
Ty1 LTR
Full-length Ty1
Full-length Ty1
-1
Chromosome II
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Observation 3 Gradual loss of signal towards
telomeres
1
log2(ratio)
0
-1
1
log2(ratio)
0
-1
Full sequence of chromosome II
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Observation 4 Aneuploidies
Chr.
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Caveat 3 Dilution effect
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Possible observation 1 Non-reciprocal
translocations?
Dunham et al., PNAS (2002)
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Possible observation 2 Single-nucleotide
changes?
Probes on the chip
Genomic DNA
0
5
10
15
20
30
35
40
45
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Gresham et al., Science (2006)
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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)!

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Acknowledgements

• Microarray group
• Karin Zueckert-Gaudenz
• Allison Peak
• Chris Seidel

• Rong Li lab
• Giulia Rancati
• Rong Li