Nucleic Acids Research 2004 32(Web Server Issue):W441-W444; doi:10.1093/nar/gkh397
© 2004, the authors
Nucleic Acids Research, Vol. 32, Web Server issue © Oxford University Press 2004; all rights reserved
KARMA: a web server application for comparing and annotating heterogeneous microarray platforms
Kei-Hoi Cheung1,2,*,
Janet Hager3,4,
Deyun Pan1,
Ranjana Srivastava6,
Shrikant Mane3,4,
Yuli Li1,
Perry Miller1,5 and
Kenneth R. Williams3,4
1 Center for Medical Informatics, Department of Anesthesiology, 2 Department of Genetics, 3 W. M. Keck Biotechnology Resource Laboratory, 4 Department of Molecular Biophysics and Biochemistry, 5 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA and 6 Celera, Rockville, MD, USA
* To whom correspondence should be addressed. Tel: +1 203 737 5783; Fax: +1 203 737 5708; Email: kei.cheung{at}yale.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received February 14, 2004; Revised and Accepted March 24, 2004
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ABSTRACT
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We have developed a universal web server application (KARMA)
that allows comparison and annotation of user-defined pairs
of microarray platforms based on diverse types of genome annotation
data (across different species) collected from multiple sources.
The application is an effective tool for diverse microarray
platforms, including arrays that are provided by (i) the Keck
Microarray Resource at Yale, (ii) commercially available Affymetrix
GeneChips® and spotted arrays and (iii) custom arrays made
by individual academics. The tool provides a web interface that
allows users to input pairs of test files that represent diverse
array platforms for either single or multiple species. The program
dynamically identifies analogous DNA fragments spotted or synthesized
on multiple microarray platforms based on the following types
of information: (i) NCBI-Unigene identifiers, if the platforms
being compared are within the same species or (ii) NCBI-Homologene
data, if they are cross-species. The single-species comparison
is implemented based on set operations: intersection, union
and difference. Other forms of retrievable annotation data,
including LocusLink, SwissProt and Gene Ontology (GO), are collected
from multiple remote sites and stored in an integrated fashion
using an Oracle database. The KARMA database, which is updated
periodically, is available on line at the following URL:
http://ymd.med.yale.edu/karma/cgi-bin/karma.pl.
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INTRODUCTION
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As microarray technology has become increasingly used by academic
researchers worldwide, the plethora of both commercial and academic
arrays available to the research community has led to a need
for a ‘universal’ tool for annotation and comparison
of array platform content. This need arises both at the inception
stage of microarray experimental design and subsequently, after
array choice and experimental implementation, for annotation
and mining of final gene expression data subsets. Additionally,
annotation of gene lists is an integral part of preparing platform
descriptions for web-posting of published data via NCBI's Gene
Expression Omnibus (GEO) (
1).
With advances in DNA sequencing technologies, the genomes of many organisms (including the human genome) have been sequenced completely. Many experimental and computational approaches have been used to extract functional information encoded in these complete sequences. As a result, a large amount and a wide variety of genome annotation data have been generated. Such annotation data are available through numerous (web-accessible) databases, including GenBank (2), LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/), Unigene (http://www.ncbi.nlm.nih.gov/), Gene Ontology (GO) (3) and SwissProt (4).
Genome-wide data analysis (e.g. microarray gene expression analysis) typically requires integration of diverse types of annotation data. Integrating these data and keeping them up to date is a significant informatics effort since these datasets evolve rapidly and are available in heterogeneous formats (including text, XML and database formats). Efforts including SOURCE (5), DRAGON (6), and Unchip (http://unchip.org:8080/bio/unchip) have been underway to provide users with timely access to such integrated data. In general, they provide a web interface for users to supply a list of gene identifiers (e.g. GenBank accession numbers), and the interface will return annotation data for each gene. We have created an integrated gene annotation database (KARMA—Keck ARray Manager and Annotator) and an associated web interface which allow users to compare and annotate their own array platforms (or simple lists of gene identifiers) as well as commonly used platforms made available through the Keck Microarray and Affymetrix Resources at Yale (http://keck.med.yale.edu/dna_arrays.htm).
The multiplicity of microarray platforms—manufactured by commercial vendors (e.g. Affymetrix GeneChips®, Agilent, MWG and ClonTech) or spotted by individual laboratories at different universities or research institutes using commercially available material (e.g. Operon, Compugen, MWG and Research Genetics) or academically generatedtargets—necessitates comparison and annotation of these platforms. By doing so, researchers are able to choose the microarray platform that best matches their needs. Resources including DRAGON, SOURCE and Unchip allow the user to submit a set of the identifiers (e.g. GenBank accession numbers) of the DNA elements laid down on chips/arrays and then return the latest annotation describing those elements. However, to perform comparison between chips/arrays based on such annotation, the user has to develop their own individual method. ARROGANT (7) allows Unigene-based comparison of two gene collections corresponding to two different microarray platforms, but it does not support cross-species comparison. RESOURCERER (8) allows comparison of different platforms involving a single or multiple species, but it can only be applied to a set of commonly used array platforms and not to a unique user-uploaded platform. In other words, it does not support comparison of custom array platforms. To address these issues, KARMA was designed to allow comparisons to be made across diverse microarray platforms (including both standard and custom-designed arrays) and also between different species using homologene information obtained from NCBI (http://www.ncbi.nlm.nih.gov/HomoloGene/). Homologous genes (based on sequence homology) do not necessarily have homologous function. The arrays to be compared may include both old and new versions (e.g. different versions of Affymetrix GeneChips), and the comparison is done based on the same version of annotation and homologene information collected by our system.
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IMPLEMENTATION
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The annotation data and homologene data are stored in an Oracle
database. We adopted the source code (a set of Perl programs)
distributed by Stanford's SOURCE, which integrates information
from multiple data sources including SwissProt, GO and NCBI's
databases including LocusLink and Unigene. The scripts load
the integrated data into the Oracle database. Unigene Cluster
IDs were used for performing comparison among platforms involving
a single species. We have extended the GO scripts to incorporate
annotation data for more species including Arabidopsis. In addition,
we have written a Perl program to fetch the homologene dataset
from the NCBI site and put the data in KARMA. Such data enable
identification of homologous sequences spotted on different
array platforms involving multiple organisms. Our web interface
was implemented in Perl/CGI, interfacing with Apache web server
running on a Linux PC.
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APPLICATIONS AND ILLUSTRATION
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Currently the KARMA tool is being used for the following types
of applications.
- To annotate individual whole arrays based on GenBank accession numbers. The flexibility of the database allows any spreadsheet text file to be uploaded from a local browser for annotation by users creating their own custom arrays or using commercial platforms.
- To compare array content in pairwise fashion to aid in the platform decision-making process during experimental design.
- To query array content for genes of interest based on GenBank accession number.
- To annotate subsets of gene expression microarray data based on GenBank accession numbers.
- To sort annotation results based on GO ID, GenBank accession numbers, SwissProt, etc.
- To create expanded annotated datasets for GEO Platform submission and provide links to existing GEO Platform files.
Figure 1 shows a web page
that allows a user to select Keck Microarray Resource array
platforms or upload their own data files for annotation and
comparison. In this case, the user wants to make a comparison
between the Affymetrix human GeneChip (HU133A) and an oligo
human spotted array. Currently, our program accepts only GenBank
accession numbers as the spot IDs, but additional types of ID
will be added. When users upload their own files, they need
to enter the position of the column (column number) that contains
the GenBank accession numbers. As described previously, KARMA
allows both single- and multiple-species comparison. When two
datasets (set A and set B) are compared for a single species,
four set comparison operations are available, namely, A
B (intersection
or common elements between the two sets), A
B (union or merging
the non-redundant elements from the sets) and two set difference
operations: A–B (elements that are in A but not in B)
and B–A (elements that are in B but not in A). For datasets
involving different species, there is only one type of comparison,
namely, identification of homologous sequences (this is similar
to the notion of intersection). Instead of comparing the whole
datasets, our system allows comparison of subsets based on user-defined
restriction conditions. Currently, we allow users to restrict
the comparison by GenBank accession numbers, gene symbols, names
or Unigene Cluster IDs. As shown in
Figure 1, the comparison
is restricted by gene names that contain the phrase ‘growth
factor’. In addition to comparison, KARMA provides up-to-date
annotation including gene symbols, functional descriptions,
Locus Link ID and Gene Ontology IDs, which can be selected for
display in the output. An option is available to sort the output
by Unigene ID, GO ID or SwissProt ID. Currently, the program
is designed to run in batch mode. In other words, the user does
not need to wait for the program to finish. When the processing
is done, the URL links to the output report files in HTML, Excel
and text formats are sent to the email address entered by the
user.
Figure 2 shows a portion of the HTML-formatted comparison/annotation
report corresponding to the input parameters as shown in
Figure 1.
As shown in
Figure 2, the GO terms (as well as the GO IDs)
associated with each entry are separated into three categories:
biological process, cellular component and molecular function.
This makes the output more informative for further analysis.
In addition, the advantage of the HTML format is that it allows
hypertext links for accessing more detailed information available
at other websites (e.g. clicking on a GO ID will return the
corresponding GO functional description). The Excel output format
also allows the hypertext links to be preserved. If the user
saves the results in text format, they can be re-uploaded to
the server for comparing with another array (either Keck, commercial
or custom). This will allow the user to compare more than two
arrays.
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Figure 1. The KARMA web interface through which datasets can be selected or uploaded for comparison and annotation.
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FUTURE DIRECTION
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In the ever-expanding informatics domain faced by biological
investigators, KARMA is a tool that filters, queries, compares
and provides annotation in a universal manner. KARMA now forms
an expandable backbone to which other database tools may be
linked, such as transcription factor and sequence databases
including AGRIS (
http://arabidopsis.med.ohio-state.edu/) and
TFSearch (
http://siriusb.umdnj.edu:18080/EZRetrieve/multi_t.jsp).
In the future, links from MAC— Microarray Convoluter (
9),
a tool for generating convoluted array format from array source
plates—and from YMD (Yale Microarray Database) (
10) query
output could be implemented. Additionally GEO Platform file
creation in soft format, available now at YMD, may be linked
to KARMA to generate annotation information for GEO Platform
descriptions. As the number and nature of databases and analytic
tools expand, so will the interoperability of KARMA with these
databases and tools.
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Notes
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The online version of this article has been published under
an open access model. Users are entitled to use, reproduce,
disseminate, or display the open access version of this article
provided that: the original authorship is properly and fully
attributed; the Journal and Oxford University Press are attributed
as the original place of publication with the correct citation
details given; if an article is subsequently reproduced or disseminated
not in its entirety but only in part or as a derivative work
this must be clearly indicated.
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REFERENCES
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