Nucleic Acids Research Advance Access published online on November 11, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp919
© Published by Oxford University Press 2009.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
MicrobesOnline: an integrated portal for comparative and functional genomics
Paramvir S. Dehal1,2,*,
Marcin P. Joachimiak1,2,
Morgan N. Price1,2,
John T. Bates1,2,3,
Jason K. Baumohl1,2,
Dylan Chivian1,2,3,
Greg D. Friedland1,2,3,
Katherine H. Huang1,2,
Keith Keller1,2,
Pavel S. Novichkov1,2,
Inna L. Dubchak1,2,
Eric J. Alm1,4 and
Adam P. Arkin1,2,3,5
1Virtual Institute for Microbial Stress and Survival, 2Lawrence Berkeley National Laboratory, Berkeley, CA 94720, 3DOE Joint BioEnergy Institute, 4Department of Biological Engineering and Department of Civil Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, 5Department of Biological Engineering and 6Department of Bioengineering, University of California, Berkeley, CA, 94720, USA
*To whom correspondence should be addressed. Tel: +1 510 643 3722; Email: psdehal{at}lbl.gov
Received September 18, 2009. Accepted October 7, 2009.
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ABSTRACT
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Since 2003, MicrobesOnline (
http://www.microbesonline.org) has
been providing a community resource for comparative and functional
genome analysis. The portal includes over 1000 complete genomes
of bacteria, archaea and fungi and thousands of expression microarrays
from diverse organisms ranging from model organisms such as
Escherichia coli and
Saccharomyces cerevisiae to environmental
microbes such as
Desulfovibrio vulgaris and
Shewanella oneidensis.
To assist in annotating genes and in reconstructing their evolutionary
history, MicrobesOnline includes a comparative genome browser
based on phylogenetic trees for every gene family as well as
a species tree. To identify co-regulated genes, MicrobesOnline
can search for genes based on their expression profile, and
provides tools for identifying regulatory motifs and seeing
if they are conserved. MicrobesOnline also includes fast phylogenetic
profile searches, comparative views of metabolic pathways, operon
predictions, a workbench for sequence analysis and integration
with RegTransBase and other microbial genome resources. The
next update of MicrobesOnline will contain significant new functionality,
including comparative analysis of metagenomic sequence data.
Programmatic access to the database, along with source code
and documentation, is available at
http://microbesonline.org/programmers.html.
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INTRODUCTION
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MicrobesOnline seeks to integrate functional genomic data with
comparative genome analysis (
1) providing two unique capabilities:
(i) a phylogenetic approach to comparative genomics, including
a tree-based browser and tools for users to build their own
tress, and (ii) microarray expression data integration. Selecting
an organism or gene of interest in MicrobesOnline leads to information
about and data viewers for experiments conducted on that organism
and involving that gene or gene product. It is possible to view
microarray data from multiple conditions as an interactive heatmap
and to analyze correlations between gene expression results
from different experiments. Among the major new features is
the ability to search the microarray data compendium for genes
with gene expression profiles similar to a query expression
profile (either based on a gene or set of genes). These new
compendium-wide functionalities allow the user to observe patterns
in gene expression changes across multiple conditions and genes,
and to search for similarities to these patterns. The information
integration and analysis performed by MicrobesOnline serves
not only to generate insights into the gene expression responses
and their regulation in these microorganisms, but also to document
experiments, allow contextual access to experimental data and
facilitate the planning of future experiments. MicrobesOnline
is actively incorporating publicly available functional genomics
data from published research, so as to centralize data on microbial
physiology and ecology in a unified comparative functional genomic
framework.
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DATA SOURCES AND ANALYSIS PIPELINE
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Sequences and annotations of bacterial and archaea genomes,
plasmids and viruses are imported from the NCBI RefSeq database
(
2). Additionally, sequences not yet deposited in RefSeq are
also loaded into MicrobesOnline by request of users. All sequences
are associated with their taxonomic lineage through mapping
to the NCBI taxonomy database (
3). Across all sequences (chromosomes,
scaffolds, contigs, genes, proteins and RNAs), identifiers are
maintained in order to link to other NCBI resources, such as
PubMed, and to facilitate cross-links to other resources, such
as IMG (
4) and RegTransbase (
5). For user-provided genomes and
for incomplete genomes downloaded from a genome center, gene
predictions are taken from the user or predicted by combining
results from CRITICA (
6) and GLIMMER3 (
7); in either case, these
predictions are superseded by the RefSeq predictions when they
become available. CRISPR and RNA loci potentially missing from
the RefSeq annotations are computed using PILER-CR (
8), CRT
(
9), tRNAScan (
10) and BLASTn.
Functional annotations of protein-coding genes include gene name, gene family, domain architecture, Enzyme Commission (EC) number (11), Gene Ontology (GO) terms (12) and user annotations. The gene name is provided by RefSeq, but all other annotations are inferred by MicrobesOnline using homology searches. FastBLAST and FastHMM (13) are used to search against the TIGRFAM (14), Superfamily (15), SMART (16), Pfam (17), Panther (18), PirSF (19), Gene3D (20), COG (21), PDB (22) and UniProt (23) databases. Characterized genes—genes that UniProt or RegTransBase link to papers (except genome papers)—are automatically highlighted on many MicrobesOnline pages. EC is from KEGG, GO from InterPro. Genes that link to UniProt papers (but not genome papers) or to RegTransBase papers are considered characterized, and are highlighted in many of our pages.
Using FastBLAST, we are also able to calculate all-against-all homology relationships for the entire microbial proteome. These results allow users to quickly find a list of homologs for any gene. Additionally, these shared regions of homology and each conserved domain alignment are used to create phylogenetic trees using the FastTree program.
Within each gene tree, MicrobesOnline identifies clades that are (mostly) present just once per genome. These are likely to be functional orthologs, but not necessarily evolutionary orthologs. MicrobesOnline uses these single-copy clades to compute MicrobesOnline ortholog groups, which are groups of likely functionally orthologous genes that are stored in the database. MicrobesOnline also computes higher quality ‘tree orthologs’ for a given query gene on demand. For details, see http://www.microbesonline.org/ortholog.html.
Gene expression data has been collected from primarily three sources, the DOE VIMSS, M3D (24) and NCBI GEO (25) database. Additionally, individual users can submit microarray data in order to use the MicrobesOnline analysis tools. MicrobesOnline contains only a partial import of the GEO database, but this will be expanded in future updates to MicrobesOnline.
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BULK DOWNLOADS AND REMOTE ACCESS
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We provide various methods for programmatic access to our data
and code. For raw database access, we provide a public MySQL
server that contains a version of MicrobesOnline with all private
genomes removed. This database can be accessed using any MySQL
compliant client or programmatic access through the standard
MySQL libraries. Information on how to use the MySQL database,
a detailed database schema description, the BLAST and FastBLAST
databases and Perl libraries and source code can be found on
the MicrobesOnline for Programmers page at
http://www.microbesonline.org/programmers.html.
Instructions for making contributions to the MicrobesOnline
code base can also be found on the MicrobesOnline for Programmers
page.
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UPDATE AND DATA SUBMISSION PROCEDURES
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MicrobesOnline has the capacity to host genomes and microarray
expression data submitted by users. These data can be kept private
through a user-defined access control list until publication
or the submitter wishes to release the data publicly. MicrobesOnline
accepts genomic data in any state whether finished sequence
or draft sequence and annotated sequence or unannotated. Instructions
for preparing the data can be found on at
http://www.microbesonline.org/privateGenomeHosting.html.
MicrobesOnline can also host private microarray data; however,
this must be done by contacting
help{at}microbesonline.org for
detailed instructions.
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MicrobesOnline FEATURE HIGHLIGHTS
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MicrobesOnline provides a comprehensive set of interfaces and
tools for comparative and functional genomics (
Table 1). These
tools perform operations on combinations of genomes, genes,
predicted gene functions, gene expression data and metabolites.
A detailed tutorial on the use of these tools is provided on
the MicrobesOnline website (
http://www.MicrobesOnline.org).
Below we highlight three of the more novel aspects of the MicrobesOnline
website: a phylogenetic tree based genome browser, a microarray
expression profile search tool and a comparative browser for
metabolic compound and pathways.
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PHYLOGENETIC TreeBrowser
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The phylogenetic TreeBrowser allows users to view a selected
gene within its evolutionary context (
Figure 1). Viewing genes
within a phylogenetic tree is not only a more biologically meaningful,
but concisely summarizes the thousands of pair-wise alignments
from all against all BLAST results into a single hypothesis
of homology. The TreeBrowser has three viewing modes, a gene
context view, a species tree view and a domains view. In the
gene context viewing mode, for any selected gene, the tree browser
displays a phylogenetic tree for a selected gene along with
genomic context of that gene and its close relatives in the
gene tree. These trees are based on a user-selectable multiple
sequence alignment that ranges from the full gene sequence to
a known domain families or
ad hoc BLAST-based clusters. The
genome context, the neighboring upstream and downstream genes
are shown colored by the gene family. This allows the user to
quickly scan the tree to determine conserved genomic context.
Because of the relatively large size of microbial gene families,
which can be over 100 000 for the largest families, it is necessary
to collapse branches and show only a single representative for
each clade. The TreeBrowser will collapse the children of a
node based on a user-selectable percent identity and pick a
single representative for the clade. The TreeBrowser highlights
characterized homologs and selects them as representatives of
the clusters.
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Figure 1. TreeBrowser in gene centric view and species view. (A) The TreeBrowser gene centric view for the E. coli K12 gene purR. The purR gene is shown centered in the browser with the neighboring up- and downstream genes. The phylogenetic tree for the purR gene is shown on the left, with a single representative selected for each clade. (B) The species view for the purR gene. The gene tree is shown on the left and the species tree on the right. Gene tree leaves are labeled numerically and these labels correspond to the comma separated list next to the names on the species tree, indicating which genes are possessed by the species, or species group, in the species tree. In this tree, we can see that the species tree indicates that the purR and rbsR are paralogs of an ancestral duplication event. Additionally, we can see that horizontal gene transfer is a likely explanation for the presence of the rpsR gene in the clade labeled Shewanella—because the gene is present in only one genome out of the 15 species represented by that clade, the could either be one HGT event or multiple independent deletion events.
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In the species tree mode, the TreeBrowser displays a phylogenetic
tree of selected gene side by side with a species tree containing
all species found in the gene tree. This species tree mode allows
users to manually reconcile the gene tree with the species tree
in order to determine events such as, horizontal gene transfer,
timing of gene duplications, orthology/paralogy relationships
or differential gene loss across lineages. Clades representing
one or more genes on the gene tree are labeled numerically and
these labels correspond to the labels on the species tree. The
names on the species tree are colored green if one or more genes
present from that species are in the gene tree, red if there
are no genes and orange if the species group has some members
with a copy of the gene.
In the domains view, the TreeBrowser displays a view similar to the gene context view. The domain architecture of the selected gene is shown and the user can select trees based on each of these domains. This view allows users to quickly scan the members of the gene family and determine if they have shared domain architecture. Additionally, the user can view the trees associated with each of the domains of the selected gene to find evidence for gene fusions or domain shuffling.
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MICROARRAY EXPRESSION PROFILING
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MicrobesOnline has a wide array of tools for analyzing microarray
gene expression data. The Experiment Browser allows user to
search for experiments in selected genomes and/or involving
specific conditions. Detailed reports on experiments are shown
in the Experiment Viewer. These reports include up- and down-regulated
genes, plots to assess array quality, experimental conditions
and expression changes by gene functional classifications. Additionally,
a Gene Expression Viewer allows users to find gene- and operon-centric
views. Detailed descriptions and instructions for their use
can be found on the MicrobesOnline website. One of the major
uses for this microarray expression data is identifying genes
which share a similar expression profile.
The gene expression profile search tool operates in two modes, a gene list view and a profile heatmap view. In both modes, the tool allows the user to select one or more genes, then calculates an average gene expression profile across the compendium of expression data and then searches for genes with a similar expression profile. In the gene list view mode, the tool returns a table of genes in descending order of their Pearson correlation coefficient to the selected gene(s) profile. The results include the correlation coefficient, gene name, gene description and buttons to either add the gene to the profile or add the gene to the analysis cart. By adding the matching gene(s) to the profile, the user can iteratively refine the profile. The profile heatmap view is a graphical heatmap representation of the selected gene(s) expression levels and those of genes identified to be similar across the microarray experiment data. The first row in the heatmap represents the input profile and genes with similar profiles are shown underneath in descending order of their Pearson correlation coefficient. The columns of the heatmap correspond to the experiments and are grouped together if the arrays are part of a series such as a time course or varying levels of a stress. The color of the cells reflects the log2 ratio value and the scale is shown to the right of the heatmap.
Results of the gene expression profile search tool can be used to identify potentially, co-regulated genes and regulatory motifs using the Motif Search tool. By adding the genes to the Gene Cart and selecting the motif search, users can potentially identify the regulatory motif.
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METABOLITES AND METABOLIC PATHWAYS
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Clicking on compounds in the KEGG pathway, browser now displays
a MicrobesOnline internal compound page. This page typically
displays the compound’s image, mass, formula and structure,
along with links to external databases (including KEGG) in which
this compound appears. MicrobesOnline also generates a series
of tables outlining the reactions in which this compound participates,
delineated by whether or not the reaction occurs in the organisms
under investigation. The table contains links to the reactions’
EC numbers, the KEGG pathway map in which the reactions occur
and the compound pages of other participants in the reactions.
The compounds participating in the reactions are color-coded
based on their KEGG RPAIR role (e.g. cofactors). Similarly,
the gene browser now has a reactions tab that lists the pathway
maps and reactions for a locus associated with the locus under
investigation.
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FUTURE PLANS
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MicrobesOnline will continue to expand in terms of data analysis
and data types supported. The next release of MicrobesOnline
will include support for metagenomes, more eukaryotic genomes
and an expanded set of tools for dealing with metabolites and
metabolic pathways. Metagenomic analysis will feature phylogenetic
trees of all genes from metagenomes and sequenced genomes and
gene content-based comparisons of metagenomic samples.
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FUNDING
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US Department of Energy Genomics: GTL program (grant DE-AC02-05CH11231).
Funding for open access charge: DOE grant.
Conflict of interest statement. None declared.
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Footnotes
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The authors wish it to be known that, in their opinion the first
and the ninth author should be regarded as joint First Authors.
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ABSTRACT
INTRODUCTION