Transcript Affymetrix Gene Chip Procedure

Affymetrix GeneChip Data Analysis
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Chip concepts and array design
Improving intensity estimation from probe pairs level
Clustering
Motif discovering and preliminary result
How is Affymetrix Gene Chip made?
Affymetrix Gene Chip Procedure
• Target preparation
fragmented
Isolate mRNA cDNA  cRNA 35~200 base fragments
(12-15ug)
biotinylated
• Target Hybridization:
200ml cocktail  probe array chip  hybridization
(16 hours at 40~60°C)
• Washing and Staining
• Probe array scan, Data analysis, and Interpretation
Concepts of Array Design
PM to maximize hybridization
MM to ascertain the degree of cross-hybridization
PM
MM
Probe pair
Probe set
Signal Intensity Estimation
• Absolute analysis
average difference
(PM-MM) / pairs used
• Comparison analysis
experimental subtract baseline average difference
• Probe pairs variation
probe pairs outlier (cross-hybridization)
arrays outlier (image contamination)
• Probe pairs level adjustment
Wing Wong Lab dChip: least square fitting model built from multiple chips
Affymetrix MAS 5.0: imputation
Clustering
• Affymetrix DMT
examine the SOM, are the clusters tight ?
experiment primarily with "Number of Epochs",
"Alpha_i" and "Sigma_i" parameters to reduce error.
multiple level clustering
examine filtering parameters to adjust min and max value.
(default is 20 and 20000)
• Wing H. Wong Lab dChip
from image file to outlier detection and clustering
• MIT Whitehead Institute GeneCluster
• Stanford Cluster and Treeview
two way clustering (genes and/or arrays)
find profile pattern not found in SOM clustering
Motif discovering
• Motif discovery and matching tools
Basic assumption: a cluster of co-regulated genes is regulated by the same transcription
factors and the genes of a given cluster share common regulatory motifs
Gibbs Motif Sampler
Neuwald et al. 1995
AlignACE
based on Gibbs Sampling, optimized for finding multiple motifs and for both strands
RSA-tools
motif finding among non-coding regions in cress, yeast, fly, worm
PlantCARE
search known motifs among 417 different plant transcription sites
TFSEARCH
search known motifs in vertebrate, arthropod, plant, yeast
Motif discovering result
• AlignACE motif finding result for a specific clustered genes
1.3e+01
8.6e+00
7.0e+00
6.5e+00
5.7e+00
7.9e-04
5.5e-01
5.5e-02
1.1e-02
6.8e-01
3.2e-03
4.4e-03
1.2e-03
2.2e-02
7.2e-03
153
27
159
460
538
GGT-TGGWT
GR---AGAA---G
G-G-R--G--GRA
AGATCCA
C-AA-C-AAA
1.9e+00
9.0e-02
1.5e-02
174
CWTTGGG
• Functional category
refer to MIPS or TIGR or Gene Ontology web site
• Exhaustive search result
fetch all possible patterns within a cluster
compare motif frequency among
random sampling and functional group
(applying M. Eisen’s Cluster/Treeview)
Supportive Information
• Schroeder lab (UCSD)
sequence retrieval and direct MEME motif finding
• Gene Microarray Pathway Profiler
genechip array statistical consideration
• MIT CCR HHMI Biopolymers Laboratory
• Databases and software tools for gene expression
software, databases, mail lists