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Microarray experiments. Database and Analysis Tools.

Microarray experiments. Database and Analysis Tools. Kate Milova cDNA Microarray Facility March 24, 2005. Outline. Microarray platforms and services at AECOM: cDNA Long Oligo Affymetrix Database (cDNA & Long Oligo) structure and content: Printing information Chip layout

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Microarray experiments. Database and Analysis Tools.

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  1. Microarray experiments. Database and Analysis Tools. Kate Milova cDNA Microarray Facility March 24, 2005 Kate Milova MolGen retreat March 24, 2005

  2. 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 Kate Milova MolGen retreat March 24, 2005

  3. Microarray Platforms at AECOM. Kate Milova MolGen retreat March 24, 2005

  4. How to choose a microarray platform. Kate Milova MolGen retreat March 24, 2005

  5. Before starting your microarray experiment. Kate Milova MolGen retreat March 24, 2005

  6. 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 Kate Milova MolGen retreat March 24, 2005

  7. Database for Analysis of Microarrays at AECOM. Contents. Chip layout Gene Annotation Printing Information • 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 • 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 • 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 Kate Milova MolGen retreat March 24, 2005

  8. Annotation sources: NCBI. UniGene ID  Accession UniGene UniGene ID  Blast against UniGene clusters Entrez Gene UniGene ID  Gene ID  GO ID NCBI Blast Software Blast Search Refseq & NT databases  Annotation Kate Milova MolGen retreat March 24, 2005

  9. Annotation sources: NCBI. UniGene ID  Accession UniGene UniGene ID Blast against UniGene clusters • NCBI  UniGene  UniGene ID: • 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”. NCBI Kate Milova MolGen retreat March 24, 2005

  10. Annotation sources: NCBI. Entrez Gene Unigene ID  Gene ID  GO ID 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. • Gene ID  GO ID: • For each Gene ID correspondingGene 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. Kate Milova MolGen retreat March 24, 2005

  11. 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 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. NCBI Blast Software Blast Search Refseq & NT databases Annotation Kate Milova MolGen retreat March 24, 2005

  12. Annotation Extraction Algorithm Formatted Data Database of cDNA & Long Oligo sequences Raw Data Sequences Homology search against RefSeq & NT Alignment quality check 90% 80% Kate Milova MolGen retreat March 24, 2005

  13. Annotation Extraction Algorithm Identity: < 90% > 90% Overlapping: < 80% > 80% 1st RefSeq hit: 1st NT hit: EDMUSDFMUSKULUSDETRIKENGLLCLONEJF FPROTEINRFTYROSINEMNWZMKINASEJHMIW Linguistic Filter OUT Kate Milova MolGen retreat March 24, 2005

  14. 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. Kate Milova MolGen retreat March 24, 2005

  15. cDNA Microarray Facility. Database. Kate Milova MolGen retreat March 24, 2005

  16. Database Search. Kate Milova MolGen retreat March 24, 2005

  17. Microarray Data Analysis Pipeline. Kate Milova MolGen retreat March 24, 2005

  18. Pipeline. Filtering. Kate Milova MolGen retreat March 24, 2005

  19. Pipeline. LOWESS Normalization. Kate Milova MolGen retreat March 24, 2005

  20. Pipeline. Data set Comparison. Kate Milova MolGen retreat March 24, 2005

  21. 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 • Rstatistical package • S-PLUS. Insightful Kate Milova MolGen retreat March 24, 2005

  22. 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 ! Kate Milova MolGen retreat March 24, 2005

  23. BACKUPS Kate Milova MolGen retreat March 24, 2005

  24. cDNA Microarray Facility.Services. Kate Milova MolGen retreat March 24, 2005

  25. cDNA Microarray Facility. Arrays. Kate Milova MolGen retreat March 24, 2005

  26. cDNA Microarray Facility. Publications. Kate Milova MolGen retreat March 24, 2005

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