introduction to microarray analysis and tools module a: approach to microarray … · 2005. 1....

Introduction to microarrayanalysis and tools

Module A: Approach to Microarray-based studies

Agnes Viale, Ph.D.Genomics Core lab

I- Introduction and potential applications of array platform

II- Existing platformsSpotted arraysAffymetrix GeneChipOther commercially available platforms

III- Experimental designProjectsTechnical limitationsFinancial considerations

IV- Steps involved in data analysis

V- ValidationReal time PCRNorthern Blot

1998- The Genomic Era

Genome: Gene+ Chromosome

Genomics Structural Genomics (1986)scientific discipline ofmapping, sequencing, andanalyzing genomes.

Functional Genomics (1995?)Analysis of genome functionCharacterization of the “transcriptome”and the “proteome”.

Pubmed records for Microarray

microarraymicroarray and cancer

Shena M, Shalon D, Davis RW, Brown PO.Quantitative monitoring of gene expression patterns with a complementary DNA microarray.Science. 1995 Oct 20;270(5235):467-70.(48 cDNA clones- Yeast)

Shalon D, Smith SJ, Brown PO.A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization.Genome Res. 1996 Jul;6(7):639-45.(874 cDNA clones-Yeast)

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

Publications

Microarray-The technical foundations

1975: Ed SouthernLabeled nucleic acid could be used to interrogate nucleic acidattached to solid support⇒Southern Blot

~1980: 1) Filter-based screening of clones libraries

2) Gridded libraries, stored in microtitre plates andstamped onto filters in fixed positions

Microarray- Key Innovations

1- Use of non-porous solid support (Glass)=> miniaturization=> fluorescence based detection

2- Methods for high-density spatial synthesis for oligonucleotides

Oligonucleotides array platform

Microarray- Definitions

Solid support (Glass) covered with spots of biomoleculesHigh Throughput Comparative hybridization platformMonitor thousands of genes in one single experiment

Oligonucleotides

GenomicDNA Protein

Applications : Gene expression profilingComparative Genomic Hybridization (CGH)Large scale protein-DNA interactionGenotyping…

Microarray- Definitions

Spotted/printed arrays:Deposition of biomolecules by contact or non contact

processIn-house or commercial (Agilent-Amersham-Nimblegen)

High Density Oligonucleotides or GeneChip arrays (Affymetrix)

Affymetrix85%

Agilent12%

Commercial Market (11-2003)

Lawsuits in microarray field

Potential applications

RNA analysis

Gene expression profilingSplice variant analysis

DNA analysisComparative Genomic Hybridization (CGH)Large scale protein-DNA interactionSNP analysisResequencing

Protein analysis (proteomics)Tissue microarrayEtc Etc Etc

RNA analysis

Spotted cDNA array Spotted oligonucleotides arraysGenechip (Affymetrix)

Splice variant analysis

Ex1 Ex2 Ex3I1 I2

Ex1 Ex3

One Strategy: exon junctions specific oligonucleotides

Ex1 Ex2 Ex3

Ex1 Ex2

Problem with current labeling/amplification protocols 3’ biased

Genomic DNA Analysis

Comparative genomic hybridization (CGH) array to studychromosomal abnormalities (gain/loss)

cDNA Oligo(very high density)

(1MB resolution)

Genomic DNA Analysis

1- BAC array CGH: Identification ofdeletion on chromosome 2

2- identificationof commonlydeleted region

3- Candidate gene

Promoter Analysis-CpG island array

CpG islands array Intergenicregions and 5’ ends of genes.

(Chromatin)

Trans. Factors

Shearing

Immunoprecipitation

DNA purification

ChIP on a CHIP = Chromatin immunoprecipitation

DNA Labeling

Comments1- Control: total chromatin ormock IP2- Amplification is necessarywhen working with mammaliancells

SNP and RESEQUENCING array

• Single Nucleotide Polymorphism array:

– high troughput analysis of DNA variation (100,000 SNParray)

– LD (linkage disequilibrium)

– LOH (loss of heterozygocity) studies

• => Mapping of candidate genes/markers

• Resequencing array:

– Comparative sequencing of candidate regions identified inmapping experiments

– Current version 30kb, next version 100kb

Proteomics

• Antibodies array

• Protein array

• Two-D gels

• High throughput mass spectrometry analysis

Tissue microarray

Used in molecular classification of cancers for validation ofgene array data by immonuhistochemistry on hundreds oftissues

Normal Tumor

I- Introdution and potential applications of array platform

II- Existing platformsSpotted arraysAffymetrix GeneChipOther commercially available platforms

IV- Steps involved in data analysistoo many steps to write

V- ValidationReal time PCRNorthern BlotRNAse protection assay

Platforms

OligonucleotidesGenechips(Affymetrix)

Spotted Arrays(cDNA or oligo)

AdvantagesReliabilityReproducibilityDisadvantagesFlexibilityCost

AdvantagesFlexibilityCostDisadvantagesReliabilityReproducibility

Platforms

Spotted array platform

cDNA clones (>10,000)

Miniprep

PCR cleaning

Quality control on gel

Printing on glass slides

•1st generation: 1998-2001

Liquid handlingstation

Miniprep

PCR cleaning

•Semi-automated•Results archivedin database

Miniprep

PCR cleaning

ArrayerPinsSlides

Plate of cDNA

Printing process

1- The Pins•Contact arrayers(Home made or commercial)

•Non-contact arrayers Ink-jet printers Piezoelectric

Split pin Microspotting pin

Pin and ring

Glasscapillary

Piezoelectriccrystal

Printing process

1- The Pins 2- The plate +cDNA

3- Slides (on a tray)

Printing process

Spotting problems

Hybridization problems

Miniprep

PCR cleaning

Oligonucleotides libraries(25 to 100 mers)

•2nd generation : 2001-….

Multi step processRequires high level of QCTIME CONSUMING

CY5 CY3

Sample A Sample B

SampleA specific/enriched

Sample B specific /enriched

Equal levels of expression

Spotted array : Basic principle (competitive hybridization)Competitive hybridization

Reverse transcriptionLabeling

Image Analysis-Gridding process

Fold change

Intensity Background

HEADER SPOT GRID ROW COL CH1I CH1B CH1AB CH2I CH2B CH2ABREMARK SOFTWARE ScanAlyzeREMARK SOFTVERS 2 . 4 4REMARK CH1 IMAGE nm2_34obfwtlC1REMARK CH2 IMAGE nm2_34obfwtlC2REMARK GRID FILE F:\Agnes\Grid\nm2_34.SAGREMARK DATE 4 / 1 8 / 0 0REMARK TIME 7:24:55 PMSPOT 1 1 1 1 2 6 8 2 1 6 2 4 9 9 4 7 4 8 8SPOT 2 1 1 2 2 0 2 2 1 5 2 5 0 9 2 7 7 9 1SPOT 3 1 1 3 2 8 6 2 1 7 2 4 8 1 5 2 7 5 9 3SPOT 4 1 1 4 1 7 0 3 2 1 1 2 6 3 1 3 5 7 7 3 1 0 4SPOT 5 1 1 5 1 3 2 4 2 1 2 2 5 9 1 0 2 5 7 5 1 0 4SPOT 6 1 1 6 2 2 4 1 2 2 1 2 5 0 1 5 6 5 8 0 9 4SPOT 7 1 1 7 2 5 7 9 2 2 2 3 0 1 2 0 4 9 8 2 1 3 3SPOT 8 1 1 8 3 8 3 2 1 7 3 0 0 1 9 5 8 2 1 3 2SPOT 9 1 1 9 1 1 3 5 2 0 9 2 6 0 7 5 7 7 8 9 8SPOT 1 0 1 1 1 0 9 6 0 2 1 7 2 8 4 6 5 9 8 3 1 0 8SPOT 1 1 1 1 1 1 1 7 8 7 2 0 7 2 8 5 1 7 5 3 8 3 1 2 0SPOT 1 2 1 1 1 2 4 8 6 9 2 0 8 2 9 7 2 6 3 5 7 8 1 2 4

Cy3 Cy5

Fluorescence intensities are proportional to level of expression of a given gene in each conditionSpot 12: Cy3/Cy5= 4869/2635= 1.8 => up regulation of 1.8 fold for gene 12 in condition A

Scatter plot

Cy5 signal (Sample A)

Platforms

Affymetrix GeneChip

5’ 3’600bp

11 Perfect Match (PM) oligonucleotides11 MisMatch (MM) oligonucleotidesMM => negative control

One PM+MM= probe pair11 PM=MM= probe setOligo = 25 mers

Affy array manufacturing- Photolithographic process

Affymetrix Genechips

Non-competitive hybridization

Sample Labeling

T7 promoter

AAAAAAAAAA

AAAAATotal RNA

RT with OdT-T7

In Vitro TranscriptionT7 RNA polymerase

Labeling: BiotinilatednucleotidesAmplification (400-1000)

Labeled cRNA

Hybridization

Image Analysis

GriddingQuantification

Complicated statisticalmysterious controversial

calculations

One number per probe set(.CHP file)

Level of expression of agene is proportional to theIntensity of fluorescence

Technology improvements

Reduction of feature size => more genes/arrayCurrent version of human array: 44,000 transcripts /array

1997-2003 2003-?

18 µm(500,000 features/array)

11 µm(1,100,000 features/array)

<5 µm(5,000,000 features/array)

Whole genome arrayIn 96 wells format

Data comparability

• Human Affy arrays

1995HuFL6800 genes

1998U95 setA,B,C,D,E arrays63,000 genes

2001U133setA,B arrays44,000 transcripts

2003U133 2.0

•Redesign the oligos•Change probe set names

•Keep same name•Change the manufacturingprocess

•Redesign the oligos•Change probe set names

⇒Difficult to compare data from one version to the next.⇒If possible : use one version of the array across one project

Other commercial platforms

• Agilent– Inkjet spotted arrays. In situ synthesis of

oligonucleotides (whole genome on 1 array)

• Nimblegen– 85,000 oligo. In situ light modulated oligo

synthesis (Digital micromirror Device or DMD).Until recently, not sold in US

• Amersham Codelink– Slides are coated with a 3-D surface chemistry

comprised of a long-chain, hydrophilicpolymer containing amine-reactive groups

=> Higher sensitivity

I- Introdution and potential applications of array platformII- Existing platforms

Spotted arraysAffymetrix GeneChipOther commercially available platforms

IV- Steps involved in data analysistoo many steps to write

Experimental Design

Ref: Microarrays for an integrative genomics. (Kohane, Kho, Butte)Design and analysis of comparative microarrayexperiments (Yang, Speed)

When to use microarray?

• Basic research: “Fishing” experiments

Not to TEST hypotheses but to GENERATE hypotheses

Comparison between two (or more) states

Study of new system: Knock Out mouse; drug treatment etc

• Molecular classification of cancer

Gene expression profiling provide an alternative molecular diagnostic

Better diagnostic, prognostic => more appropriate treatment

Identification of markers => elucidation of molecular mechanismsunderlying diseases.

Experimental design

Choices you have to make

Parameters which will determine your choices

Replicates1 Platform 2

Budget4 Amount of RNA 3

Replicates

Biological replicates: YES

Array 1

Array 2

Array 3

Fake replicates: NO

Should I do replicates?

HoxA9_GFP(August2003) HoxA9-2

HoxA9-3

22181all genes

How many replicates?

Statistician’s answer: 5Realistic answer: 3Investigator’s choice: 1 or 2

RNA Quality

Degraded (or partially degraded RNA) = bad array data•Wrong conclusions•Waste your time chasing false positives

•Homogeneity in a sample set is VERY IMPORTANT

RNA quantity

1- Enough RNA to process without amplification1 microgram < RNA

2- Not enough RNA : linear amplification20 ng< RNA < 700ng

Double strand cDNA

Total RNART

IVTcRNA

Double strand cDNA

RNA quantity

3- ?< RNA<20 ng:

Double strand cDNA

Total RNART

IVTcRNA

Double strand cDNA

Labeled cRNA

Laser Capture microdissectionFACS

Pooling vs. Amplification

RNA=300 ng

One array

0.9µg => no amplification

POOLING

No statistical power

2XAmplificationfor each sample

3 arrays

“Some statisticalPower”

Financial considerations

Statistician’s answer: 5Realistic answer: 3Investigator’s choice: 1 or 2

Why are the answers different depending on who is answeringthe question about the number of replicates?

Affy arrays (New York state) $ 400/ whole genome (human mouse rat)Labeling (average in US)= $250 => $650/samplesTwo conditions (KO and WT), in triplicates = $3,900

Expensive technology but with good experimental design, itprovides data that cannot be generated in other ways and is worththe investment.

Experimental design-Conclusions

1. Right questions and right controls

2. Homogeneity among samples

3. Don’t be cheap

For a mathematical/statistical approach of experimental design:•Microarrays for an integrative genomics. (Kohane, Kho, Butte)•Design and analysis of comparative microarrayexperiments (Yang, Speed)

I- Introdution and potential applications of array platformII- Existing platformsIII- Experimental design

IV- Steps involved in data analysisData set QCNormalizationFeature (gene) filteringReplicate analysisClusteringStatistical testsPathway

Data set QCGeneChip built-in control 1: % present genes

Varies according to tissue (30 to 60%)

Higher on last generation of arrays (more sensitive)

Allows easy identification of outliers in a dataset2x amplification

Control Genes Signal(3'/5')

ArrayType Filename

Factor Background

HUMGAPDH/

M33197

HSAC07/X003

51HG-U133A JS_U133A_SetD-A-.CHP 2.42 3.222 Avg: 61.23 10210 45.80% 1.60 38.46

HG-U133A JS_U133A_SetD-A+.CHP 2.50 3.240 Avg: 60.43 10000 44.90% 1.92 40.90HG-U133A JS_U133A_SetD-B-.CHP 2.44 4.643 Avg: 65.01 9404 42.20% 1.84 53.10

HG-U133A JS_U133A_SetD-B+.CHP 2.74 2.603 Avg: 73.12 10165 45.60% 1.65 45.89HG-U133A JS_U133A_SetD-C-.CHP 2.72 2.157 Avg: 65.20 10211 45.80% 1.79 53.34

HG-U133A JS_U133A_SetD-C+.CHP 2.89 2.255 Avg: 73.21 10006 44.90% 1.73 50.72HG-U133A JS_U133A_SetD-D+.CHP 2.77 2.820 Avg: 71.02 9767 43.80% 1.97 52.87

HG-U133A JS_U133A_SetD-E-.CHP 2.28 5.662 Avg: 54.54 8686 39.00% 2.29 77.90HG-U133A JS_U133A_SetD-E+.CHP 3.10 3.300 Avg: 83.54 9156 41.10% 1.55 36.78

3X amplification

HG-U133A MLe_U133A_C1.CHP 2.33 10.221 Avg: 55.61 8140 36.50% 10.39 391.75HG-U133A MLe_U133A_C2.CHP 2.43 5.836 Avg: 58.10 8626 38.70% 13.54 109.31

HG-U133A MLe_U133A_C3.CHP 2.16 14.664 Avg: 51.92 6435 28.90% 5.48 132.42HG-U133A MLe_U133A_S1.CHP 2.37 6.432 Avg: 59.89 7220 32.40% 4.00 171.67

HG-U133A MLe_U133A_S2.CHP 2.23 9.504 Avg: 53.16 7371 33.10% 6.49 156.78HG-U133A MLe_U133A_S3.CHP 2.39 13.135 Avg: 60.31 4267 19.10% 5.10 62.62

Test3 Test3_MLe_Mario-0.5.CHP 1.80 5.644 Avg: 55.19 84 26.90% 5.98 73.67Test3 Test3_MLe_Mario-1.CHP 2.18 5.074 Avg: 68.98 85 27.20% 3.76 64.38

Number Present

Data set QC

GeneChip built-in control 2: 3’/5’ratio for “house keeping” genes

Housekeeping Controls:Probe Set Sig(5') Det(5') Sig(M') Det(M') Sig(3') Det(3') Sig(3'/5')HUMISGF3A/M97935 66.7 P 374.9 P 658.9 P 9.88HUMRGE/M10098 455.6 P 244 P 577.2 P 1.27GAPDH 24262.7 P 19308 P 19918.3 P 0.82b-ACTIN 14621.8 P 18055 P 15733 P 1.08M27830 494.8 P 1132 P 150.4 A 0.3

5’ 3’5’ M 3’

Sample Name

Percent Present Sig(5') Det(5') Sig(M') Det(M') Sig(3') Det(3') Sig(all) Sig(3'/5')

s1 39.40% GAPDH 216.00 P 352.50 A 1880.40 P 816.30 8.71B-ACTIN 63.00 A 537.70 A 7356.60 P 2652.42 116.80

s2 44.10% GAPDH 211.30 P 544.40 P 3047.90 P 1267.87 14.42B-ACTIN 42.20 A 539.10 P 7630.10 P 2737.13 180.95

s3 41.40% GAPDH 189.10 P 747.30 P 6559.80 P 2498.71 34.70B-ACTIN 102.50 A 248.00 A 13449.20 P 4599.90 131.16

1<Signal 3’ probe/Signal 5’ probe<3

Genes filtering

WT KO ResFiltering parameters

Fold change cut-offMerge Replicate analysisP-values (statistical test)

Advice 1: Most stringent filtering

Advice 2: Do not forget about biology

Less stringent

Replicate analysis

Exp: WT vs. KO- Triplicate for each conditionList of regulated genes?Filtering on fold change (and Affy p-val- next module)

3 comparisons

9 comparisons

Replicate analysis

Exp: WT vs. KO- Triplicate for each conditionList of regulated genes?Filtering on fold change (and Affy p-val- next module)

3 comparisons

If samples were processed two by two, then you should matchEach KO with the corresponding WT

Replicate Analysis

“9 comparisons” more stringent analysis than “3 comparisons”“9 comparisons” included in the “3 comparisons”

225 104 0 2 1 0 4 11 0

ClusteringClustering = grouping genes (or samples) with similar expressionpattern.

Why clustering?

Groups of Genes Which Share Common Patterns ofExpression May Share Common Transcriptional Regulation

Diagnostic ALL BM samples (n=327)

3σ-3σ -2σ -1σ 0 1σ 2σσ = std deviation from mean

TEL-AML1BCR-ABL

Hyperdiploid >50E2A-PBX1

MLL T-ALL Novel

HierarchicalPartitional

K-meansSOM (Self Organizing Map)PCA (Principal Component Analysis)

Supervised clustering

Clustering

Principle

Real life situation: Signal to noise ratio issue

+Noise

Nick Socci, Bioinformatics Core, MSKCC

Clustering

• Not always necessary

• No straightforward solution for clusteringa. Choose an algorithm which make the fewest/simplest

assumptions.b. At least know the assumptions the algorithm is making.c. Supervised vs unsupervised: may use an unsupervised

algorithm but features selection supervised: may not be a badidea to start.

• Interactive process between the person who will analyze the dataand the biologist. This interaction should start at the experimentaldesign level

Nick Socci, Bioinformatics Core, MSKCC

Statistical Tests

To test the null hypothesis : the hypothesis for each gene is that there isno difference in the mean gene expression intensities in the groupstested

=> a gene will have equal means across every group

• Rejection of the null hypothesis (i.e. acceptance of the alternativehypothesis) indicates that the means intensities are from twodifferent populations.

• The value(s) returned by ttest is the P-value: indicates the probabilityof getting a mean difference between the groups as high as what isobserved by chance. The lower the P-value, the more significant thedifference between the groups.

Statistical Tests

Kruskal-Wallistest

Wilcoxon-Mann Whitney

Non Parametric

Welch ANOVAWelch T-testParametric

(variance not equal)

ANOVAStudent’s T-test

Parametric

(variances equal)

More than 2groups

2 groups

Recommended formost cases

When to use what?

Least assumptionMore than 5 replicatesper group

To use if very fewreplicates or groupwithout replicates

Pathway analysisA pathway is a graphical representation of the interaction betweengene products in a biological system.

Genes can be superimposed on the pathway, allowing you to view theirexpression levels in a biological context

Pathway analysis

•GenMapp (http://www.genmapp.org/) (human, mouse, rat,yeast)

•EASE: the Expression Analysis Systematic Explorer(http://david.niaid.nih.gov/david/ease.htm)

•Cytoscape (http://www.cytoscape.org)

•Pathway Processorhttp://cgr.harvard.edu/cavalieri/pp.html (yeast, B. subtilis)

(Module: Functional interpretation of high-throughput data)

Data analysis-Conclusions

• Data QC => eliminate outliers samples

• Gene filtering => Help focusing on “interesting” genes

• Clustering => only if necessary

=> noise-dependant

• Statistical test => use the appropriate one

• Pathway analysis => Global picture of regulation

I- Introdution and potential applications of array platformII- Existing platformsIII- Experimental design

IV- Steps involved in data analysisData set QCNormalizationFeature (gene) filteringReplicate analysisClusteringStatistical testsPathway

V- ValidationReal time PCRNorthern BlotRNAse protection assayISH

Validation of microarray data

• Do I need to validate my data?

– YES

• Why?

– Because it will make you feel more confident about your results

– Because the reviewers will ask for it

• How?

Oct.2004 survey: Do you validate your microarray data? If yes, how?Response Percent

Northern Analysis 31.60%

RNase Protection 8.40%

Real-time PCR 87.40%

In-situ hybridization 16.80%

None 9.50%

Other 8.40%

9 5Total Respondents

Northern-blot: RNA> 5µg

WT Ob WT Ob WT Ob WT Ob

WT Ob WT Ob WT Ob

~31.5 ~9.8 8.2 ~5.4 3.2

~-7.7 -7.7 -6.1 -4.5 -3.1

1* || 778 -126* || 420 147 || 1314 61* || 436 1587 || 5143

548 || 71* 5986 || 776 3579 || 724 8411 || 1837 743 || 237

WT Ob WT Ob

Average Difference (wt || ob)

Fold Change

Average Difference (wt || ob)Fold Change

Cyclophilin

Soukas et Al.Genes Dev. 2000 Apr 15;14(8):963-80.

Real Time PCR

• “Real time quantitative PCR.”Genome Res. 1996 Oct;6(10):986-94 Heid CA, Stevens J, Livak KJ, Williams PM.

• Very “popular”• Request smaller amounts of RNA

•Real-time reverse-transcriptase (RT) PCR quantitates the initialamount of the template most specifically, sensitively andreproducibly, and is a preferable alternative to other forms ofquantitative RT-PCR, which detects the amount of final amplifiedproduct.•Based on the detection and quantification of a fluorescent reporter

Real Time PCR

Expensive assays:TaqmanMolecular beaconScorpions

Cheaper alternativeSYBR-green : measures the amplicon production

(including non-specific amplification and primer- dimercomplex)

Q-PCR -2.3 -3.0

Array -2.9 -4.1

6hr 24hr

Q-PCR -1.5

Array -1.8

Real Time PCR

Internal reference:All data should be “normalized” to a reference gene (HPRT, Actin, GAPDH, Cyclophilin…)

Correlation between array data and Q-PCR data: Good to excellent even at fold changes<2

CONCLUSIONS

•Everyone should take advantage of this HYPOTHESESGENERATING Technology, ideal to “explore” a new biologicalsystem

•Expensive technology but with good experimental design, itprovides data that cannot be generated in other ways

Thousands of daysThousands of experiments=> Study of one gene

One dayOne experiment⇒Study thousands of genes

Before microarray After microarray

introduction to microarray analysis and tools module a: approach to microarray … · 2005. 1....

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