Gene Expression: Microarray Theory and NCBI GeneCentered Resources

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Gene Expression Microarray Theoryand NCBI
Gene-Centered Resources
June 20, 2007
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Eucaryote Gene Expression Control
nucleus
cytosol
inactive mRNA
mRNA degradation control
Primary RNA transcript
DNA
mRNA
mRNA
RNA transport control
translation control
transcriptional control
RNA processing control
protein
protein activity control
nucleus membrane
Expressed Genes mRNA
inactive protein
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Gene expression profiles
Expression (relatively levels to reference point
at 0)
Time/Condition
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Goal of Microarray Experiments

• Regulation/function in pathway/cellular
  state/phenotype
• Disease diagnosis / disease gene identification

Gene expression
Microarray data
Biological pathway
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Similarity between Profiles
expression

• Similarity measure
• Euclidean distance
• Correlation coefficient
• Trend
• Correlation coefficient
• often works better.

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time
Expression profile
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Data-Mining through Clustering

• Assumptions for clustering analysis
• Expression level of a gene reflects the genes
  activity.
• Genes involved in same biological process exhibit
  
• statistical relationship in their expression
  profiles.

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Idea of Clustering

• Clustering group objects into clusters so that
• objects in each cluster have similar features
• objects of different clusters have dissimilar
  features

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Methods of Clustering

• discriminant analysis (Fisher,1931)

• K-means (Lloyd,1948)

• hierarchical clustering

• self-organizing maps (Kohonen, 1980)

• support vector machines (Vapnik, 1985)

• single linkage (dendrogram)

• minimal spanning tree based clustering

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Issues in Cluster Analysis

• A lot of clustering algorithms
• A lot of distance/similarity metrics
• Which clustering algorithm runs faster and uses
  less memory?
• How many clusters after all?
• Are the clusters stable?
• Are the clusters meaningful?

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Which Clustering Method Should I Use?

• What is the biological question?
• Do I have a preconceived notion of how many
  clusters there should be?
• How strict do I want to be? Spilt or Join?
• Can a gene be in multiple clusters?
• Hard or soft boundaries between clusters

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Clustering Algorithms

• K-Means clustering (most popular)
• Hierarchical clustering
• Minimum spanning tree-based clustering

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NCBI Databases and Services

• GenBank largest sequence database
• Free public access to biomedical literature
• PubMed free Medline
• PubMed Central full text online access
• Entrez integrated molecular and literature
  databases
• BLAST highest volume sequence search service
• VAST structure similarity searches
• Software and Databases

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Types of Databases

• Primary Databases
• Original submissions by experimentalists
• Content controlled by the submitter
• Examples GenBank, SNP, GEO, Probe, GenSat
• Derivative Databases
• Built from primary data
• Content controlled by third party (NCBI)
• Examples Refseq, TPA, RefSNP, UniGene, NCBI
  Protein, Structure, Conserved Domain, Gene

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Gene-centered Databases

• Sequences
• GenBank
• Reference Sequences
• Expression
• UniGene
• GEO
• Probe
• GenSat

• Variation (dbSNP)
• HomoloGene
• Gene

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What is GenBank? NCBIs Primary Sequence Database

• Nucleotide only sequence database
• Archival in nature
• Historical
• Reflective of submitter point of view
  (subjective)
• Redundant
• GenBank Data
• Direct submissions (traditional records)
• Batch submissions (EST, GSS, STS)
• ftp accounts (genome data)
• Three collaborating databases
• GenBank
• DNA Database of Japan (DDBJ)
• European Molecular Biology Laboratory (EMBL)
  Database

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RefSeq NCBIs Derivative Sequence Database

• Curated transcripts and proteins (NM_, NP_)
• reviewed
• human, mouse, rat, fruit fly, zebrafish,
  arabidopsis
• microbial genomes (proteins), and more
• Model transcripts and proteins (XM_, XP_)
• Assembled Genomic Regions (contigs) (NT_, NW_)
• human
• mouse
• rat
• Chromosome records (NC_)
• Human genome
• microbial
• organelle

• chicken
• honeybee
• sea urchin

• zebrafish
• cow
• dog

• black poplar

srcdb_refseqProperties
ftp//ftp.ncbi.nih.gov/refseq/release/
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GenBank to RefSeq Transcript (NM_)
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RefSeqs Annotation Reagents
Genomic DNA (NC, NT, NW)
Scanning....
Model mRNA (XM) (XR)
Model protein (XP)
?
Curated mRNA (NM) (NR)
Curated Protein (NP)
RefSeq
GenBank Sequences
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Mouse Assembly
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Expressed Sequences

• UniGene
• GEO
• Probe
• GenSat

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Expressed Sequence Tags in Entrez
Total 41 million records Human 7.9
million Mouse 4.7 million Cow 1.3
million Rice 1.2 million Zebrafish 1.2
million Maize 1.2 million Xenopus
tropicalis 1.0 million Rat 0.9
million Wheat 0.9 million Chicken 0.6
million Barley 0.4 million
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What is UniGene?
A gene-oriented view of sequence entries

• MegaBlast based automated sequence clustering
• Now informed by genome hits New!
• Nonredundant set of gene oriented clusters
• Each cluster a unique gene
• Information on tissue types and map locations
• Includes known genes and uncharacterized ESTs
• Useful for gene discovery and selection of
  mapping reagents

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EST hits Human mRNA
Albumin mRNA
5 EST hits
3 EST hits
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UniGene
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Gene Catalog X. laevis MLH1Cluster
Uncharacterized ESTs
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Associating Sequences Human ALB Cluster
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Expression Data
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Gene Expression Omnibus

• Expression data repository
• Primary GEO data records
• Platform (e.g. Affymetrics Gene Chip, Expression
  Set 230 Array )
• Sample (e.g. E19 type II epithelial cells, 16h,
  control )
• Series (e.g. lung development, fetal type II
  epithelial cells, strain response, microarray, 6
  samples)
• Derivative GEO Databases
• Datasets
• Biologically and statistically compatible Samples
  curated and suitable for GEO analysis tools
• Profiles
• Expression measurements for an individual gene
  across all Samples in a DataSet.

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Experiment Types in GEO

• Array-based
• Single Channel
• Dual Channel
• Antibody arrays
• Universal Microarray System (UMAS)
• Genomic (Comparative Genomic Hybridization (CGH),
  Chromatin ImmunoPrecipitation (ChIP-chips),
  tiling arrays, SNP detection)
• Non-array-based
• Serial Analysis of Gene Expression
• Massively Parallel Signal Sequencing
• Serial Analysis of Ribosomal Sequencing Tags
• Mass Spectrometry Proteomics Data

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Expression Platform
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Expression Platform
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Expression Sample
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Expression Series
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GEO Dataset
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Expression ProfileGAD1
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Entrez Probe

• Nucleic acid reagents
• Sequence targeted oligos
• Over 7 million entries
• Applications
• Genotyping
• Gene Silencing
• SNP Discovery
• Genome Mapping
• Gene Expression
• Real time PCR
• In Situ hybridization

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Ataxia Telangiaectasia Mutated Gene Probes
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Resequencing Amplicon SNP Discovery
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Gene Silencing Probes
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Gene Expresion Probe to Gensat
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Gene Expression Nervous System Atlas

• NINDS and Rockefeller University
• BAC Transgenic Mouse Strains
• Enhanced GFP expression
• Driven by endogenous gene regulatory sequences
• Histologic sections mouse brain
• Confocal Microscopy GFP reporter
• Immunohistochemical staining transgenics
• In Situ Hybridization native expression
• Results Important for understanding brain
  microanatomy and development

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GENSAT Dopamine Receptor 2 (Drd2)
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Bright Field Immunostaining BAC Transgenic
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dbSNP Nucleotide Polymorphisms
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NCBIs SNP Database

• Primary Database and Derivative (RefSNP)
• Single Nucleotide Polymorphism
• Repeat polymorphisms
• Insertion-Deletion Polymorphisms
• 29 Species
• Over 46 million submissions (submitted SNPs)
• Over 26 million submissions (reference SNPs)

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Submitted SNP
Hemachromatosis SNP
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RefSNP
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RefSnp Analysis GeneView
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Mapping to Structure
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Population Genetics Genotype
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The Gene Database

• Gene Centered Information
• Unifies LocusLink and microbial Genomes
• 2.4 million records for 3,822 taxa

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Genes MLH1 One Stop Shopping
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Genes MLH1 One Stop Shopping (cont.)
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Genes Display Options and Links
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Homologene

• Completely Annotated Eukaryotic Genomes
• Homologous UniGene determined for other organisms
• Protein similarities first
• Guided by taxonomic tree
• Includes orthologs and paralogs
• Provides Neighboring function for Entrez Gene
  database

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Homologene Cluster
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WWWAccess
Entrez BLAST
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Entrez Database Integration
Word weight
PubMed abstracts
3-D Structure
3 -D Structure
Taxonomy
VAST
Genomes
Phylogeny
Neighbors Related Structures
Protein sequences
Nucleotide sequences
BLAST
BLAST
Neighbors Related Sequences BLink Domains
Neighbors Related Sequences
Hard Link
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Entrez Use Gene for everything
BLink
Homologene Gene Neighbors
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Glutamate decarboxylases

• Finding
• Sequences
• Expression Information
• Molecular Probes
• Homologs in other species
• Genomic Context
• Polymorphisms
• Structures and Conserved Domains

www.ncbi.nlm.nih.gov
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Mouse GAD Genes
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GAD2 Gene Record
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GAD2 Sequences
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Gene Table Genomic Sequences
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The Map Viewer Genomic Sequences
Download data here
Zoom or enter region of interest
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Expression Data
GENSAT
GEO
UniGene
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GenSat BAC transgenics
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GEO Profiles
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Preview / Index Refining Results
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Gad2 Expression Profile
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Genes with similar expression
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Molecular Probes
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RSA Primers (Human)
Known SNPs in Red
Download entire tiling set
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Finding Homologs HomoloGene
Protien mRNA Genomic
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HomoloGene Cluster
Gene Links
Protein Links
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Homologous UniGene
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UniGene Hs.231829 GAD2
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Expression Profile
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Finding Homologs 2 BLink
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BLink BLAST Link (Best Hits)
Redundant Proteins
First 200 only
Goldfish homolog
BLAST
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Finding Polymorphisms Human
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SNP GeneView
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Related Structures structure model
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Sequence Similar Structures
Conserved Domain
Link to Structure
Link to Alignment
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Pig DOPA decarboxylase
Structure Neighbors
Cn3D viewer
3D Domain Neighbors
Conserved Domains
Pubchem compound
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Alignment Based Model
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Better Model Conserved Domain
Ala-Gly from dbSNP
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Service Addresses

• info_at_ncbi.nlm.nih.gov
• blast-help_at_ncbi.nlm.nih.gov