carlo colantuoni & rafael irizarry april 19, 2006 [email protected] gene annotation in genomics...
TRANSCRIPT
Carlo Colantuoni&
Rafael Irizarry
April 19, 2006
Gene Annotation in Genomics Experiments With a Focus on Tools in BioConductor
Biological Setup
Every cell in the human body contains the entire human genome: 3.3 Gb or ~30K genes.
The investigation of gene expression is meaningful because different cells, in different environments, doing different jobs express different genes.
Tasks necessary for gene expression analysis:
Define what a gene is.
Identify genes in a sea of genomic DNA where <3% of DNA is contained in genes.
Design and implement probes that will effectively assay expression of ALL (most? many?) genes simultaneously. Cross-reference these probes.
Cellular Biology, Gene Expression, and Microarray Analysis
DNA
RNA
Protein
AAAAA
Gene: Protein coding unit of genomic DNA with an mRNA intermediate.
START STOPprotein coding
5’ UTR 3’ UTR
mRNA
GenomicDNA 3.3 Gb
DNAProbe
~30K genes
Sequence is a Necessity
From Genomic DNA to mRNA Transcripts
EXONS INTRONS
RNA editing & SNPs
Alternative splicingAlternative start & stop sites in same RNA molecule
~30K
>30K
Transcript coverage Homology to other transcripts
Hybridization dynamics 3’ bias
Protein-coding genes are not easy to find - gene density is low, and exons are interrupted by introns.
Sequence Quality!
Redundancy!
Completeness?
Unsurpassed as source of expressed sequence
Chaos?!?
From Genomic DNA to mRNA Transcripts
~30K
>30K
>>30K
Transcript-BasedGene-Centered Information
Possible mis-referencing:Genomic GenBank Acc.#’sReferenced ID has more NT’s than probeOld DB buildsDB or table errors – copying and pasting 30K rows in excel …
Using RefSeq’s can help.
Design of Gene Expression Probes
Content: UniGene, Incyte, Celera Expressed vs. Genomic
Source: cDNA libraries, clone collections, oligos
Cross-referencing of array probes (across platforms):
Sequence <> GenBank <> UniGene <> HomoloGene
From Genomic DNA to mRNA Transcripts
From Genomic DNA to mRNA Transcripts
http://www.ncbi.nlm.nih.gov/Entrez/
Functional Annotation of Lists of Genes
KEGGPFAM
SWISS-PROTGO
DRAGONDAVID
BioConductor
Analysis of Functional Gene Groups
•One of the largest challenges in analyzing genomic data is associating the experimental data with the available metadata, e.g. sequence, gene annotation, chromosomal maps, literature.
•The annotate and AnnBuilder packages provides some tools for carrying this out.
•Using AnnBuilder. It is possible to build associations with specific gene lists, eg. hgu95a package for Affymetrix HGU95A GeneChips.
•The annotate package maps to GenBank accession number, LocusLink LocusID, gene symbol, gene name, UniGene cluster, chromosome, cytoband, physical distance (bp), orientation, Gene Ontology Consortium (GO), PubMed PMID.
Analysis of Functional Gene Groups
Functional Gene/Protein Networks
DIPBINDMINTHPRD
PubGenePredicted Protein Interactions
Analysis of Gene Networks
9606 is the Taxonomy ID for Homo Sapiens
Predicted Human Protein Interactions
Predicted Human Protein Interactions
Used high-throughput protein interaction experiments from fly, worm, and yeast to predict human protein interactions.
Human protein interaction is predicted if both proteins in an interaction pair from other organism have high sequence homology to human proteins.
>70K Hs interactions predicted>6K Hs genes
Analysis of Gene Networks
Carlo ColantuoniClinical Brain Disorders Branch, NIMH, NIH
Dept. Biostatistics, [email protected]
Thanks to …
Rafael Irizarry
Scott Zeger
Jonathan Pevsner
http://www.ncbi.nlm.nih.govhttp://www.ncbi.nlm.nih.gov/Entrez/http://www.ncbi.nih.gov/Genbank/http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotidehttp://www.ncbi.nlm.nih.gov/dbEST/http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Proteinhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genehttp://www.ncbi.nlm.nih.gov/LocusLink/http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigenehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologenehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMedhttp://www.ncbi.nlm.nih.gov/PubMed/http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cddhttp://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtmlhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snphttp://www.ncbi.nlm.nih.gov/SNP/http://eutils.ncbi.nlm.nih.gov/entrez/query/static/advancedentrez.html http://www.ncbi.nlm.nih.gov/geo/http://www.ncbi.nlm.nih.gov/RefSeq/
FTP:ftp://ftp.ncbi.nlm.nih.gov/ftp://ftp.ncbi.nlm.nih.gov/repository/UniGeneftp://ftp.ncbi.nih.gov/pub/HomoloGene/
NCBI Web Links
NUCLEOTIDE:
http://genome.ucsc.edu/
http://www.embl-heidelberg.de/
http://www.ensembl.org/
http://www.ebi.ac.uk/
http://www.gdb.org/
http://bioinfo.weizmann.ac.il/cards/index.htmlhttp://www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl
PATHWAYS and NETWORKS:
http://www.genome.ad.jp/kegg/
ftp://ftp.genome.ad.jp/pub/kegg/ (http://www.genome.ad.jp/anonftp/)
http://dip.doe-mbi.ucla.edu
http://dip.doe-mbi.ucla.edu/dip/Download.cgi
http://www.blueprint.org/bind/
http://www.blueprint.org/bind/bind_downloads.html
http://160.80.34.4/mint/index.php
http://160.80.34.4/mint/release/main.php
http://www.hprd.org/
http://www.hprd.org/FAQ?selectedtab=DOWNLOAD+REQUESTS
http://www.pubgene.org/ (also .com)
PROTEIN:
http://us.expasy.org/
ftp://us.expasy.org/
http://www.sanger.ac.uk/Software/Pfam/
http://www.sanger.ac.uk/Software/Pfam/ftp.shtml
http://smart.embl-heidelberg.de/
http://www.ebi.ac.uk/interpro/
http://us.expasy.org/prosite/
ftp://us.expasy.org/databases/prosite/
More Web Links
http://www.bioconductor.org/http://apps1.niaid.nih.gov/david/http://www.geneontology.org/http://discover.nci.nih.gov/gominer/index.jsphttp://pubmatrix.grc.nia.nih.gov/http://pevsnerlab.kennedykrieger.org/dragon.htm
SAVAGE:
Detection of More Subtle Functionally Related Groups
of Gene Expression Changes
EXP#1
Swiss-Prot
30KPFAM
KEGG
~3K
10K
~40K annotations
DRAGON SAVAGE
Differential Expression of FunctionalGene Groups within One Experiment
EXP#4EXP#3EXP#2EXP#1BioDB
Differential Expression of a Single FunctionalGene Group Across Multiple Experiments
DRAGON
SAVAGE
Similar Differential Expression Patterns Across Multiple Experiments
p value
0.0
<0.1
ALL
CN
CN
CN
CN
The distribution of gene expression values for each gene group in each sample is plotted as a single point in low dimensional space. This is achieved using Principal Components Analysis along with Non-Metric Multi-Dimensional Scaling.
1
1
EX
P#1
EX
P#1
2
2
EX
P#2
EX
P#2
5
4
3
5
4
3
X
CN
X
PING:
Detection of Differential Expression in Functional
Networks of Proteins
Interaction Networks in Gene Expression Data
Large Protein Interaction Network
Network Regulated in Sample #1
Network Regulated in Sample #1
Network Regulated in Sample #2
Large Protein Interaction Network
Network Regulated in Sample #1
Network Regulated in Sample #2
Network Regulated in Sample #3
Large Protein Interaction Network
Networkof Interest
Network Regulated in Sample #1
Network Regulated in Sample #2
Network Regulated in Sample #3
Large Protein Interaction Network
PING
1
10
100
1000
10000
100000N
T's
in G
en
Ban
k (m
illio
ns)
1984 1994 2004
Genomic DNA Content
1. Interspersed repeats (~1/2 Hs. genome)2. (Processed) pseudogenes3. Simple sequence repeats4. Segmental duplications (~5% Hs. genome)5. Blocks of tandem repeats (can be very large)6. Genes: Promoters - Exons – Introns <3%
defining what a gene is - protein coding unit of genomic DNA with an mRNA intermediateidentifying genes within genomic DNA
protein-coding genes (mRNA)functional RNA genes - tRNA, rRNA, snoRNA, snRNA, miRNA
prokaryotes eukaryotes
AAAAA
Gene: Protein coding unit of genomic DNA with an mRNA intermediate.
START STOPprotein coding
5’ UTR 3’ UTR
mRNA
GenomicDNA 3.3 Gb
Protein
AAAAA
Gene: Protein coding unit of genomic DNA with an mRNA intermediate.
START STOPprotein coding
5’ UTR 3’ UTR
mRNA
GenomicDNA 3.3 Gb
Protein
~30K genes
Sequence is a Necessity
How is a gene definedin “wet” biology and in silico?
Seq. from mRNA sample
Seq. on array
Array probe design:
Source – cDNA libraries, oligos, clone collections
Content – UniGene, Celera, Incyte
Transcript coverage
Homology to other transcripts
Hybridization dynamics – hyper-multiplex hyb rxn
Empirical validation
3’ bias
Alt. splicing - known and not
Alt. start / stop site in same RNA molecule
Less important: RNA editing, SNPs
Cross-referencing of array probes:GenBank <> UniGene <> HomoloGene
Possible mis-referencing:Genomic GenBank Acc.#’sReferenced ID has more NT’s than probeOld DB buildsDB or table errors
Finding genes in eukaryotic DNA
ORF identification – Three Letter Genetic Code (codons) 4*4*4. It is possible to translate any stretch of genomic DNA into protein, but that doesn’t mean we have identified a protein coding gene!
There are several kinds of exons:-- non-coding-- initial coding exons-- internal exons-- terminal exons-- some single-exon genes are intronless
What We Are Going To Cover
Cells, Genes, Transcripts –> Genomics Experiments
Sequence Knowledge Behind Genomics Experiments
Annotation of Genes in Genomics Experiments