Tiling Arrays

Madelaine GogolProgrammer AnalystMicroarraymcm@stowers-institute.org

Norman PavelkaPostdocRong Li Labnxp@stowers-institute.org

Technology and Methods SeminarMarch 29, 2007

http://wiki/research/MadelaineGogol

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?

What is a Tiling Array?

Gene 1

Probe 1

A microarray with many probes distributed in an evenly spaced way across an entire genome.

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

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

ChIP-chip

PCR w/aa-dNTPHybridize to Microarray

Analyze image, calculate ratios

array CGH

Hybridize to Microarray

In-house yeast tiling array (YOGie)

• Covers the yeast genome

• Just printed

• resolution ~ 250 bases

• freely available

Spotted Microarray Manufacturing

Operon Probe Set

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

Intergenic Probe Set: Design

• Design target: yeast Intergenic regions• original goal: leave no area > 500 uncovered

Gene 1 Gene 2

Array Oligo

Selector (AOS)

Fasta format 140-mer sequences tiling the intergenic regions

9,405 70-mer sequences from the intergenic regions

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)

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

Gene 1

operon probe

3’5’

Array Oligo

Selector (AOS)

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

Gene 1

operon probe

3’5’

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)

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

Agilent Microarray Manufacturing

Agilent Tiling Arrays

ChIP-on-chip Arabidopsis Whole Genome C. elegans Whole GenomeDrosophila Whole GenomeHuman CpG Island Human ENCODE 244KHuman 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 244KHuman Genome 105KHuman Genome 44KMouse Genome 244KMouse Genome 105KMouse Genome 44K

244k

105k

44k

15k

1

2

4

8

$400

$640

$720

$800

$320

$180

$100

per slide

Agilent Tiling Arrays: formats and cost

per hyb

$400

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

earray

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

Affymetrix Microarray Manufacturing

http://www.affymetrix.com/support/technical/byproduct.affx?cat=exparrays

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.

Summary of Tiling Arrays (only yeast shown)

Type #spots per array

probe size

resolution per slide cost

per array cost

customizable?

Homemade YOGie arrays

26,000 70 250 between

0 0 some effort

Agilent 44K 44,000 (4) 60 160 between

$720 $180 easily

Agilent 244K 244,000 60 overlap by 18 $400 $400 easily

Affy 3,200,000(pm/mm)

25 overlap by 20 $500 $500 no

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

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

Data Visualization: Genome Browsers: UCSC

Data Visualization: Genome Browsers: IGB

http://wiki/research/IntegratedGenomeBrowser

Data Analysis: Sliding windows

ChIPOTle, PeakFinder, R scripts, etc.

Data Analysis: Annotating and comparing peaks

http://main.g2.bx.psu.edu/

Data Analysis: Average Gene analysis

Profile of binding across an average gene

0 20 40 60 80

0.0

0.2

0.4

0.6

AcH4/WT

positions in average gene and surrounding intergenic regions

ave

rag

ed

exp

ress

ion

ra

tios

http://wiki/research/Microarray/AverageGeneAnalysis

Summary

• Tiling arrays– CHip-chip, CGH, expression

• Which ones are available– In-house, Agilent, Affy

• Data analysis

• Future...

The Future of Tiling Arrays

The resolution continues to increase...

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

5th floorBuilding 2

N

Bioinformatics

Microarray

Allison Karin Brian ChrisMe

Thanks!

Microarray

Bioinformatics

All the labs that use microarrays!

Bing LiWorkman lab

Jennifer BuppJasperson lab

Norman PavelkaRong Li Lab

Technology & Methods Technology & Methods SeminarSeminar

““Adventures in Electron Adventures in Electron Microscopy”Microscopy”

Rhonda AllenRhonda AllenHistologyHistology

Thursday, April 26th, 1:00 p.m.Thursday, April 26th, 1:00 p.m.ClassroomClassroom

(1(1stst floor, Administration Building) floor, Administration Building)

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