introduction to microarray analysis and tools · user access, roles, security - - + barcode...
TRANSCRIPT
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Introduction to microarrayanalysis and tools
Module B: Survey of Microarray Analysis ToolsCommercial Tools
Agnes Viale, Ph.D.Genomics Core lab
MSKCC
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Microarray assay life cycle
BiologicalQuestion
SamplePreparation
MicroarrayHybridization
Microarray Detection
Data Analysis& modeling
M.Shena and R. Davis,MIcroarray biochip technology
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Plan
I- GeneChip Operating System (GCOS)
II- Genespring
III-Submission to public repository
IV- NetAffx
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Affymetrix GeneChip-Definitions
5’ 3’600bp
PMMM
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Software name
– MicroArray Suite 4.0 (MAS 4.0) = Empirical algorithm
– Microarray Suite 5.0 (MAS 5.0) = Statistical algorithm
– Genechip Operating System (GCOS)
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Data comparability
• Affy arrays
1995 1998 2001 20032.0 platform
•Redesign the oligos•Change probe set names
•Keep same name•Change the manufacturingprocess
•Redesign the oligos•Change probe set names
• Software
MAS 4.0 MAS 5.0 GCOS
•New Algorithm •Same algorithm•Different data management
GenechipSoftware
•New Algorithm
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Signal intensity
1- Genechip software: use all pairs
∑Α∈
−Α
=j
jj MMPMAvDiff )(1 A: probe pairs selected by the
software
2- MAS 4.0: excluded outlier pairs: PM-MM values that were more than 3 SDfrom the mean PM-MM value
- not robust average- negative Average difference if MM>PM
3- MAS5.0: weighted mean of avg.(PM-MM)- Probe intensities preprocessed for global background.- PM-IM intensities are log transformed- Robust mean of probe set values taken using Tukey Biweight.
)}{log(BiweightTukey *jj MMPMsignal −=
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MAS4.0/MAS5.0
MAS 5.0 MAS 4.0
Detection p-valueChange p-value
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MAS 5.0/GCOS
+--Centralized Data Sharing
+--User Access, Roles, Security
+++-Barcode Support, Automation
++-MIAME Standard Template
++-Publishing to AADM Database
++-Manage & Associate Projects,Experiments, Samples & Data
+++Gene Expression Data Analysis(Statistical Algorithm (CHP)
-++Instrument Control / Data Acquisition
GCOSServer
GCOSClient
MAS
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Raw files-comparison files
.Exp
.DAT
.CEL
.CHP
.CHP
Raw CHP file B Raw CHP file A
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GCOS
Software status window
Data window
Files window
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.Exp file
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.DAT file
DAT= scan
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Using the .DAT file
1- To identify defective arrays
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Using the .DAT file
1- To identify image problem
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.DAT file
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.CEL file
.CEL= Computerized version of the .DAT file CEL file is used to generate the .CHP file
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Raw . CHP file
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comparison . CHP file
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.RPT file
• Data set QC
GeneChip built-in control 1: % present genes
GeneChip built-in control 2: 3’/5’ratio for “house keeping” genes
Right click a .CHP file
=> Report (.RPT) file
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.RPT file
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.RPT file
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Access to probe cell information
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Generate an comparison file
- Drag and drop the experimental CEL file- Choose the baseline file- Enter the output file name
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Scatter plot
2X up
2X down
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Scatter plot
“Background box”
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Next stepsData export in ExcelData export to third party software
Applied Maths, GenExplore™ :BioDiscovery, GeneSight:GeneData AG -Expressionist. LION Bioscience AG'sMolecular Applications Group, Stingray™.MolecularWare, Inc.: ArrayAnalyzerDBPartek, Inc., Partek Pro 2000Rosetta Inpharmatics. Resolver™Scanalytics, Inc. , MicroArray SuiteSilicon Genetics' GeneSpringTMSpotfire, Inc., .Media Cybernetics, Array-Pro(R).Microarray Software developed by Stanford UniversityTIGR (The Institute for Genome Research) offers software tools (free foracademic institutions) for array analysis.
OmniViz, Inc., OmniViz Pro Xpogen Inc., PathlinX
…
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Plan
I- GeneChip Operating System (GCOS)
II- Genespring
III- NetAffx
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Plan
I- Introdution and potential applications of array platformII- Existing platformsIII- Experimental design
IV- Steps involved in data analysisData set QCNormalizationFeature (gene) filteringReplicate analysisClusteringStatistical testsPathway
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Genespring interface
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Choice of “genome”
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Data import
TXT files from a Genechiparray or from a spottedarray or from any othertype of array as long asyou have a “signal”associated with a identifier(gene, transcript, protein,other)
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Samples information
• Sample-centric system (not experiment centric)
• Sample attributes format is MIAME compliant
Minimum information about a microarray experiment (MIAME)-toward standards for microarray data.Nat Genet. 2001 Dec;29(4):365-71.
MIAME goal : to specify the minimum information that must bereported about a microarray experiment in order to ensure itsinterpretability, as well as potential verification of the results
• MIAME format required for microarray data publication
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1. Experimental design: the set of the hybridisation experiments as a whole2. Array design: each array used and each element (spot) on the array3. Samples: samples used, the extract preparation and labeling4. Hybridizations: procedures and parameters5. Measurements: images, quantitation, specifications6. Controls: types, values, specifications
Hybridisation ArraySample
Analysis
Experiment Normalisation
6 parts in MIAME
MIAME
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Samples information
• Sample-centric system (not experiment centric)
• MIAME compliant sample attributes format
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Experiment parameters
Parameters can be used for gene filtering with a statistical test
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Gene filtering
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Gene filtering
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Gene lists
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Gene lists
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Union/Intersection of gene lists
Venn Diagram
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Union/Intersection of gene lists
Venn Diagram
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Statistical analysis
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Statistical analysis output
•Venn diagram•Clustering•Pathway analysis•…
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Clustering tools in GS
Hierarchical clusteringExperiments and samples
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Projection methods
•Principal component analysis (PCA)•Multi-Dimensional Scaling (MDS)•Not clustering methods but can beused to determine or visualize clusterstructure if present
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Microarray assay life cycle
BiologicalQuestion
SamplePreparation
MicroarrayHybridization
Microarray Detection
Data Analysis& modeling
M.Shena and R. Davis,MIcroarray biochip technology
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PLAN
I- GeneChip Operating System (GCOS)
II- Genespring
III-Submission to public repository
IV- NetAffx
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Data submission to public repository
Do you submit your data to MIAME compliant microarray public database?Response % Response Total
Always 9.70% 6sometimes 19.40% 12Only if requested by publisher 38.70% 24never 33.90% 21
Total Respondents 62
Which database are you submitting your data to?Response % Response Total
GeneExpression Omnibus- (GEO-NIH) 43.50% 27Array Express (EMBL) 29% 18Other (please specify) 33.90% 21
Total Respondents 62
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Data submission to GEO
3 steps process:
1- Submission of theplatform (Array type)
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Data submission to GEO
3 steps process:
1- Submission of theplatform (Array type)
2- Submission of thesamples (MIAME)
ID_REF VALUE DETECTION Detection p-valueAFFX-MurIL2_at 13.4 A 0.953518AFFX-MurIL10_at 17.3 A 0.843268AFFX-MurIL4_at 18.1 A 0.749204AFFX-MurFAS_at 15.8 A 0.425962AFFX-BioB-5_at 730.6 P 0.001593AFFX-BioB-M_at 1952.8 P 0.000044AFFX-BioB-3_at 1267.6 P 0.000147AFFX-BioC-5_at 3155.5 P 0.00007AFFX-BioC-3_at 2296.3 P 0.000052AFFX-BioDn-5_at 2987.8 P 0.000044AFFX-BioDn-3_at 16968.8 P 0.00006AFFX-CreX-5_at 31299.5 P 0.000044AFFX-CreX-3_at 47550 P 0.000044AFFX-BioB-5_st 117.8 A 0.165861AFFX-BioB-M_st 155 A 0.108979AFFX-BioB-3_st 179.6 A 0.327079
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Data submission to GEO
3 steps process:
1- Submission of theplatform (Array type)
2- Submission of thesamples (MIAME)
3- Submission of a“serie”( experiment)
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Plan
I- GeneChip Operating System (GCOS)
II- Genespring
III-Submission to public repository
IV- NetAffx
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NetAffx
Def: comprehensive resource of functional annotations and public database
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NetAffx
Def: comprehensive resource of functional annotations and public database
Accession number
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Access to NetAffx
Free registrationUpdated every quarter
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Quick query input
Key wordGene symbolPublic DB numberProbe set name
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Quick query output
GO: Gene Ontology Pathway information
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Pathway Diagram
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Quick query output
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Detailed information
•Genechip Array Information•Probe design information•Genomic Alignment of target sequence•Public domain and Genome references•Functional annotations•Sequence
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Genechip Array Information
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Probe design/ Genomic Alignment
Link to UC Santa Cruz Genome Browser
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Public domain
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Functional Annotations
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Sequence information
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Batch query
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Batch query
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Batch query output
Export data to ExcelGene ontology brower
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Gene ontology browser
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•DNA microarrayBowtell, Sambrook, CSHL
•A Biologist's Guide to Analysis of DNA Microarray Data Steen Knudsen
•http://ihome.cuhk.edu.hk/%7Eb400559/array.html
•DNA Microarray (genome chip) Leming Shihttp://www.gene-chips.com/
Useful links and lectures
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Conclusion
Proteomics
Human Genetics(Genotyping)
ClinicalDatabase
Genomics
Basic ResearchAnimal Models of Human Cancer
Pathway Analysis
GLOBAL UNDERSTANDING OF MOLECULAR BASIS OF CANCER