Transcript milova_032405 - Microarray Facility

Microarray experiments.
Database and Analysis Tools.
Kate Milova
cDNA Microarray Facility
March 24, 2005
Kate Milova
MolGen retreat
March 24, 2005
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Outline.
 Microarray platforms and services at AECOM:
 cDNA
 Long Oligo
 Affymetrix
 Database (cDNA & Long Oligo) structure and content:
 Printing information
 Chip layout
 Annotation
 Annotation algorithms and data mining
 On-line Analysis Tools:
 Normalization
 Signal filtering
 Data sets comparison
 Statistical packages and Analysis software
 Summary
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Microarray Platforms at AECOM.
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How to choose a microarray platform.
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Before starting your microarray experiment.
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cDNA Microarray Facility. Home page.
Standart & Custom Arrays. Description & Prices
Hybridization, labeling, bioinformatics,
workshops
Database for cDNA & Long Oligo Arrays.
Analysis Pipeline
AECOM cDNA microarray facility. Supported
publications
Useful links of analysis tools
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Database for Analysis of Microarrays at
AECOM. Contents.
Printing Information
Chip layout
 Chip name
 Specie
 Number of spots
 Number of controls
 Number of pen
domains
 Number of slides
 Printing pattern
 Distance between
spots
 Number of rows
 Number of columns
 Printing date
 Master chip
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 Chip name
 Spot
information
(Accession or
clone id or
bacterial
control)
 Spot location
 Library name
 Clone location
on 384 plate
 Clone location
on 96 plate
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Gene Annotation
 Accession
 Clone ID
 Clone end
 Vector name
 Clone name
 UniGene cluster ID
 Best blast hit
 Main blast parameters
(score, E-value, %
identity, blast date, etc.)
 Gene ID
 Gene symbol
 Gene synonyms
 Chromosome
 Map location
 GO IDs
 GO Annotation
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Annotation sources: NCBI.
UniGene ID 
Accession
UniGene
NCBI
UniGene ID  Blast
against UniGene
clusters
Entrez Gene
UniGene ID 
Gene ID 
GO ID
Blast Software
Blast Search
Refseq & NT
databases 
Annotation
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Annotation sources: NCBI.
UniGene ID 
Accession
UniGene
UniGene ID  Blast
against UniGene
clusters
 NCBI  UniGene  UniGene ID:
NCBI
 UniGene ID for cDNA arrays is obtained from the
UniGene source file for each particular accession number
of the clone.
 NCBI  UniGene  Blast:
 UniGene ID for Long Oligo arrays is obtained from blast
results
 Blast search was done with the set of oligo sequences
against UniGene clusters with cutoff 99% for sequence
identity and 90% for overlapping.
 UniGene ID for the oligo hitting multiple UniGene
clusters is marked as an “Ambiguous cluster ID”.
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Annotation sources: NCBI.
 UniGene ID  Gene ID:
 All information retrieved from ‘Enrez Gene’ project is based
on the UniGene cluster ID and corresponding Gene ID.
 Gene ID is ambiguous in ‘Gene ID’ to ’UniGene cluster ID’
connection.
 Parsing filter was used to eliminate ambiguous Gene IDs.
NCBI
Entrez Gene
Unigene ID 
Gene ID 
GO ID
 Gene ID  GO ID:
 For each Gene ID corresponding Gene Ontology IDs
were retrieved from Entrez Gene source file
 There might be a few or more then 10 different GO IDs
for a Gene ID. All of them are collected.
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Annotation sources: NCBI.
 Blast Software package is installed on the
microarray server.
 This software allows to format databases and run batch
homology search for any combination of custom
databases and query sequences.
 Refseq & NT databases. Annotation
 Loaded
NCBI
formatted and periodically updated on the
microarray server.
 When databases are updated we run blast search of
cDNA and Long Oligo sequences.
Blast results are parsed using our algorithm for
annotation extraction.
Blast Software
Blast Search
Refseq & NT
databases
Annotation
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Annotation Extraction Algorithm
Sequences
Raw Data
Database of cDNA &
Long Oligo sequences
Formatted
Data
Homology search against RefSeq & NT
Alignment
90% quality
check
80%
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Annotation Extraction Algorithm
EDMUSDFMUSKULUSDETRIKENGLLCLONEJF
FPROTEINRFTYROSINEMNWZMKINASEJHMIW
Linguistic
Filter
Overlapping:
1st RefSeq hit:
1st NT hit:
< 90%
> 90%
OUT
Identity:
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< 80%
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> 80%
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Annotation sources: Gene Ontology.
Biological
process
Molecular
function
Gene
Ontology
 Gene Ontology.
 Multiple GO IDs for each
Gene ID are retrieved in the
previous step from Entrez
Gene ( if available).
Cellular
compartment
 Gene Ontology annotation for all GO IDs is kept in three
different information fields: biological processes,
molecular function and cellular compartment. For each of
the fields all available annotation was prefiltered with
redundancy check and concatenated.
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cDNA Microarray Facility. Database.
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Database Search.
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Microarray Data Analysis Pipeline.
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Pipeline. Filtering.
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Pipeline. LOWESS Normalization.
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Pipeline. Data set Comparison.
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Statistical packages and Analysis software.
Microarray Analysis Software:
 GeneTraffic: client-server systems for microarray data
analysis. Iobion
 GeneSpring: cutting-edge tools for expression analysis.
Agilent Technologies
 GeneSifter. GeneSifter
 BASE. Lund University
 Data Mining:
 PathwayAssist: Interaction Explore Software. Stratagen
 Pathways Analysis. Ingenuity
 Tools for Statistical Analysis:
 SAM: Significance Analysis of Microarrays. Stanford
 R statistical package
 S-PLUS. Insightful
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Summary
 Multiple microarray platforms are available at AECOM:
 Affymetrix
 cDNA arrays
 Long Oligo
 Custom arrays
 Data analysis and annotation
Database for Analysis of Microarrays containes all information
about our arrays, cDNA and oligo sets
 Sequences annotation is updated and integrated into the
database
 Web interface of the database makes it easy to search for a
particular gene, synonyms, map location, function, etc.
 Easy to use web based analysis pipeline – get your results in
just 5 minutes. List of ‘Up’, ‘Down’ regulated genes with full gene
annotation.
 We are here for help and consultation !
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BACKUPS
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cDNA Microarray Facility. Services.
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cDNA Microarray Facility. Arrays.
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cDNA Microarray Facility. Publications.
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