Download Diapositiva 1

Annotation, Databases,
GO, Pathways,and
all those things
Information on microarray data
• Consists of different
types of informations
– Genes annotations
– Samples annotations
– Genes expression
levels
– Covariates
– Experimental design
– etc
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Annotation: Relating
probesets to genes
Use of microarray clone
annnotation
• Often, the result of microarray data analysis is a list of
genes.
• The list has to be summarized with respect to its
biological meaning.
• For this, information about the genes and the related
proteins has to be gathered.
– If the list is small (let’s say, 1–30), this is easily done by reading
database information and/or the available literature.
– Sometimes, lists are longer (100s or even 1000s of genes).
Automatic parsing and extracting of information is needed.
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From clone information to
genes and proteins (1)
• Microarrays are produced using
information on expressed sequences as
EST clones, cDNAs, partial cDNAs etc.
• At the other end, functional information is
generated (and available) for proteins.
• Hence, there is a need to map a clone
sequence ID to a protein ID.
• This is a non-trivial task
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From clone information to genes
and proteins: a non-trivial task
• First, there are usually hundreds of ESTs (and
several cDNA sequences) that map to the same
gene.
– The Database Unigene tries to resolve this multiplicity
problem by sequence clustering.
– An alternative approach is taken by Locus Link. This
is a quite stable repository of genomic loci, supposed
to be a single gene.
– Since the emphasis is on well-characterised loci,
Locus Link is not complete.
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From clone information to genes
and proteins: multiple ways to go
• There are other projects like RefSeq (NCBI) or TIGR Gene Indices.
• According to the cross-references available for a certain microarray,
one or the other may be advantageous
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An example:
The human genome
• With the completion of the human genome sequence,
you’d think that such ambiguities can be resolved. In
fact, that is not the case.
– Part of the problem is due to the fact that it is hard to predict
gene structure (intron/exon) without knowing the entire mRNA
sequence, which happens for about two-thirds of all genes.
– Then, there are errors in the assembly (putting together the
sequence snippets). A typical symptom is that a gene appears to
map to multiple loci on the same chromosome, with very high
sequence similarity.
– But there are also sequences that are nearly indentical, but
duplicated. This has happened not long ago in evolution by
means of transposable elements.
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The human genome:
Some figures
• Currently, it’s estimated that the human genome contains about
25,000 – 30,000 genes that code for 50,000 – 100,000 different
transcripts (and thus, proteins).
• Unigene (human section) contains 105,680 clusters, but 45,999
• of them are of size 2 or less.
• RefSeq DNA contains 28,097 human sequences.
• ENSEMBL contains 21,787 predicted genes, 31,609 predicted
transcripts.
• Fully computational methods like Genscan produce more than
65,000 predictions.
• Locus Link contains 15,248 genes with known function, and further
6038 genes without function annotation
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Function annotation
• Probably, the most important thing you want to know is what the
genes or their products are concerned with, i.e. their function.
• Function annotation is difficult:
– Different people use different words for the same function, or
– may mean different things by the same word.
– The context in which a gene was found (e.g. “TGF-induced gene”) may
not be particularly associated with its function.
• Inference of function from sequence alone is error-prone and
sometimes unreliable.
• The best function annotation systems (GO,SwissProt) use human
beings who read the literature before assigning a function to a gene
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The Gene Ontology
• To overcome some of the problems, an
annotation system has been created: The Gene
Ontology (http://www.geneontology.org).
– It represents a unified, consistent system, i.e. terms
occur only once, and there is a dictionary of allowed
words.
– Furthermore, terms are related to each other: the
hierarchy goes from very general terms to very
detailed ones.
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Cross-references with GO
• The GO database exists independently
from other annotation databases
• There exist cross-references (GOA)
enabling to relate other annotations with
those contained in GO
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Bioconductor and annotations
• Annotation information is managed in Bioconductor
through metadata packages
• These packages contain one-to-one and one-to-many
mappings for frequently used chips, especially Affymetrix
• Information available includes gene names, gene
symbol, database accession numbers, Gene Ontology
function description, enzmye classification number (EC),
relations to PubMed abstracts, and others.
• There are several packages implementing functionalities
to deal with annotation information: annotate, Annbuilder,
ontoTools, GOstats and many more
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Static vs. Dynamic Annotation
Static Annotation:
• Bioconductor packages containing annotation
information that are installed locally on a
computer
• well-defined structure
• reproducible analyses
• no need for network connection
Dynamic Annotation:
• stored in a remote database
• more frequent updates  possibly different
result when repeating analyses
• more information
• one needs to know about the structure of the
database, the API of the webservice etc.
Available Metadata
• EntrezGene
is a catalog of genetic loci that connects curated sequence
information to official nomenclature. It replaced LocusLink.
• UniGene
defines sequence clusters. UniGene focuses on protein-coding
genes of the nuclear genome (excluding rRNA and
mitochondrial sequences).
• RefSeq
is a non-redundant set of transcripts and proteins of known
genes for many species, including human, mouse and rat.
• Enzyme Commission (EC)
numbers are assigned to different enzymes and linked to genes
through EntrezGene.
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Available Metadata
• Gene Ontology (GO)
is a structured vocabulary of terms describing gene products
according to molecular function, biological process, or cellular
component
• PubMed
is a service of the U.S. National Library of Medicine. PubMed
provides a rich resource of data and tools for papers in journals
related to medicine and health. While large, the data source is not
comprehensive, and not all papers have been abstracted
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Available Metadata
• OMIM
Online Mendelian Inheritance in Man is a catalog of human genes
and genetic disorders.
• NetAffx
Affymetrix’ NetAffx Analysis Center provides annotation resources
for Affymetrix GeneChip technology.
• KEGG
Kyoto Encyclopedia of Genes and Genomes; a collection of data
resources including a rich collection of pathway data.
• IntAct
Protein Interaction data, mainly derived from experiments.
• Pfam
Pfam is a large collection of multiple sequence alignments and
hidden Markov models covering manycommon protein domains17
and families.
Available Metadata
• Chromosomal Location
Genes are identified with chromosomes, and where appropriate
with strand.
• Data Archives
The NCBI coordinates the Gene Expression Omnibus (GEO);
TIGR provides the Resourcerer database, and the EBI runs
ArrayExpress.
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Annotation Packages
• An early design decision was to provide metadata on a per chiptype basis (e.g. hgu133a, hgu95av2)
• Each annotation package contains objects that provide mappings
between identifiers (genes, probes, …) and different types of
annotation data
• One can list the content of a package:
> library("hgu133a")
> ls("package:hgu133a")
[1] "hgu133a" "hgu133aACCNUM"
[3] "hgu133aCHR" "hgu133aCHRLENGTHS"
[5] "hgu133aCHRLOC" "hgu133aENTREZID"
[7] "hgu133aENZYME" "hgu133aENZYME2PROBE"
[9] "hgu133aGENENAME" "hgu133aGO"
[11] "hgu133aGO2ALLPROBES" "hgu133aGO2PROBE"
[13] "hgu133aLOCUSID" "hgu133aMAP"
[15] "hgu133aMAPCOUNTS" "hgu133aOMIM"
[17] "hgu133aORGANISM" "hgu133aPATH"
[19] "hgu133aPATH2PROBE" "hgu133aPFAM"
[21] "hgu133aPMID" "hgu133aPMID2PROBE"
[23] "hgu133aPROSITE" "hgu133aQC"
[25] "hgu133aREFSEQ" "hgu133aSUMFUNC_DEPRECATED"
[27] "hgu133aSYMBOL" "hgu133aUNIGENE"
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A little bit of history...
(the pre-SQL era)
before: hgu95av2
hgu95av2.db
now:
Annotation Packages
• Objects in annotation packages used to be environments,
hash tables for mapping  now things are stored in SQLite
DB
• Mapping only from one identifier to another, hard to reverse
• quite unflexible
• The user interface still supports many of the old
environment-specific interactions:
You can access the data directly using any of the standard
subsetting or extraction tools for environments:
get, mget, $ and [[.
> get("201473_at", hgu133aSYMBOL)
[1] "JUNB"
> mget(c("201473_at","201476_s_at"), hgu133aSYMBOL)
$`201473_at`
[1] "JUNB"
$`201476_s_at`
[1] "RRM1"
> hgu133aSYMBOL$"201473_at"
[1] "JUNB"
> hgu133aSYMBOL[["201473_at"]]
[1] "JUNB"
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Working with Metadata
Suppose we are interested in the gene BAD.
> gsyms <- unlist(as.list(hgu133aSYMBOL))
> whBAD <- grep("^BAD$", gsyms)
> gsyms[whBAD]
1861_at 209364_at
"BAD" "BAD"
> hgu133aGENENAME$"1861_at"
[1] "BCL2-antagonist of cell death"
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Working with Metadata
Find the pathways that BAD is associated with.
> BADpath <- hgu133aPATH$"1861_at"
> kegg <- mget(BADpath, KEGGPATHID2NAME)
> unlist(kegg)
01510
"Neurodegenerative Disorders"
04012
"ErbB signaling pathway"
04210
"Apoptosis"
04370
…
"Colorectal cancer"
05212
"Pancreatic cancer"
05213
"Endometrial cancer"
05215
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Working with Metadata
We can get the GeneChip probes and the unique EntrezGene loci
in each of these pathways. First, we obtain the Affymetrix IDs
> allProbes <- mget(BADpath, hgu133aPATH2PROBE)
> length(allProbes)
[1] 15
> allProbes[[1]][1:10]
[1] "206679_at" "209462_at" "203381_s_at"
"203382_s_at"
[5] "212874_at" "212883_at" "212884_x_at"
"200602_at"
[9] "211277_x_at" "214953_s_at"
> sapply(allProbes, length)
01510 04012 04210 04370 04510 04910 05030 05210
05212 05213
85 169 162 137 413 243 39 167 156 111
05215 05218 05220 05221 05223
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194 137 160 117 110
Working with Metadata
And then we can map these to their Entrez Gene values.
> getEG = function(x) unique(unlist(mget(x,
hgu133aENTREZID)))
> allEG = sapply(allProbes, getEG)
> sapply(allEG, length)
01510 04012 04210 04370 04510 04910 05030
05210 05212 05213
37 84 81 67 187 130 18 82 72 51
05215 05218 05220 05221 05223
85 68 74 53 53
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.db Packages
• Data in the new .db annotation packages is stored in
SQLite databases
 much more efficient and flexible
• old environment-style access provided by objects of class
Bimap (package AnnotationDbi)
left
object
right
object
left
object
right
object
left
object
right
object
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.db Packages
• Data in the new .db annotation packages is stored in
SQLite databases
 much more efficient and flexible
• old environment-style access provided by objects of class
Bimap (package AnnotationDbi)
left
object
left
object
right
object
name
left
object
attr1 = value1
attr2 0 value2
right
object
right
object
 bipartite graph
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DBI
• collection of classes and methods for database interaction
• they abstract the particular implementations of common
standard operations on different types of databases
• resultSet: operations are performed on the database, the user
controls how much information is returned
dbSendQuery
create result set
dbGetQuery
get all results
dbGetQuery(connection, sql query)
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.db Packages
Notice that there are a few more entries here. They give you
access to a connection to the database.
> library("hgu133a.db")
> ls("package:hgu133a.db")
[1] "hgu133aACCNUM" "hgu133aALIAS2PROBE"
[3] "hgu133aCHR" "hgu133aCHRLENGTHS"
[5] "hgu133aCHRLOC" "hgu133aENTREZID"
[7] "hgu133aENZYME" "hgu133aENZYME2PROBE"
[9] "hgu133aGENENAME" "hgu133aGO"
[11] "hgu133aGO2ALLPROBES" "hgu133aGO2PROBE"
[13] "hgu133aMAP" "hgu133aMAPCOUNTS"
[15] "hgu133aOMIM" "hgu133aORGANISM"
[17] "hgu133aPATH" "hgu133aPATH2PROBE"
[19] "hgu133aPFAM" "hgu133aPMID"
[21] "hgu133aPMID2PROBE" "hgu133aPROSITE"
[23] "hgu133aREFSEQ" "hgu133aSYMBOL"
[25] "hgu133aUNIGENE" "hgu133a_dbInfo"
[27] "hgu133a_dbconn" "hgu133a_dbfile"
[29] "hgu133a_dbschema"
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> con <- hgu133a_dbconn()
> q1 <- "select symbol from gene_info“
> head(dbGetQuery(con ,q1))
symbol
1
A2M
2
NAT1
3
NAT2
4 SERPINA3
extract information from a database table as data.frame
> toTable(hgu133aSYMBOL)[1:3,]
probe_id symbol
1 217757_at
A2M
2 214440_at
NAT1
3 206797_at
NAT2
reverse mapping
> revmap(hgu133aSYMBOL)$BAD
[1] "1861_at"
"209364_at"
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Lkeys, Rkeys: Get left and right keys of a Bimap object
> head(Lkeys(hgu133aSYMBOL))
[1] "1007_s_at" "1053_at"
"117_at"
"121_at"
"1255_g_at" "1294_at"
> head(Rkeys(hgu133aSYMBOL))
[1] "A2M"
"NAT1"
"NAT2"
"SERPINA3" "AADAC"
"AAMP"
nhit: number of hits for every left key in a Bimap object
> table(nhit(revmap(hgu133aSYMBOL)))
1
2
3
4
5
6
7
8
9
10
11
12
13
18
19
8101 2814 1273 475 205
77
3
4
1
2
1
1
19
15
5
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