Nucleic Acids Research Advance Access originally published online on May 30, 2008
Nucleic Acids Research 2008 36(Web Server issue):W149-W153; doi:10.1093/nar/gkn293
Nucleic Acids Research, 2008, Vol. 36, No. suppl_2 W149-W153
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Uprobe 2008: an online resource for universal overgo hybridization-based probe retrieval and design
Robert T. Sullivan1,
Caroline B. Morehouse1,
NISC Comparative Sequencing Program2 and
James W. Thomas1,*
1Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322 and 2Genome Technology Branch and National Institutes of Health Intramural Sequencing Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA
*To whom correspondence should be addressed. Tel: +404 727 9751; Fax: +404 727 3949; Email: jthomas{at}genetics.emory.edu
Received January 23, 2008. Revised April 17, 2008. Accepted April 29, 2008.
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ABSTRACT
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Cross-species sequence comparisons are a prominent method for
analyzing genomic DNA and an ever increasing number of species
are being selected for whole-genome sequencing. Targeted comparative
genomic sequencing is a complementary approach to whole-genome
shotgun sequencing and can produce high-quality sequence assemblies
of orthologous chromosomal regions of interest from multiple
species. Genomic libraries necessary to support targeted mapping
and sequencing projects are available for more than 90 vertebrates.
An essential step for utilizing these and other genomic libraries
for targeted mapping and sequencing is the development of the
hybridization-based probes, which are necessary to screen a
genomic library of interest. The Uprobe website (
http://uprobe.genetics.emory.edu)
provides a public online resource for identifying or designing
‘universal’ overgo-hybridization probes from conserved
sequences that can be used to efficiently screen one or more
genomic libraries from a designated group of species. Currently,
Uprobe provides the ability to search or design probes for use
in broad groups of species, including mammals and reptiles,
as well as more specific clades, including marsupials, carnivores,
rodents and nonhuman primates. In addition, Uprobe has the capability
to design custom probes from multiple-species sequence alignments
provided by the user, thus providing a general tool for targeted
comparative physical mapping.
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INTRODUCTION
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Genomic resources which were once restricted to humans and traditional
model systems, such as whole-genome sequence assemblies and
large-insert genomic libraries, are now being developed for
an ever expanding list of species. Driven in part by the demonstrated
utility of interspecies sequence comparisons for genome annotation
and decreasing costs of DNA sequencing, these new genomic tools
are providing an ever expanding platform for studies in comparative
genomics. While whole-genome shotgun sequencing has largely
replaced the clone-by-clone-based method to sequence the human
genome (
1,
2), high-quality targeted genomic sequencing of chromosomal
regions of interest derived from clone-based physical maps provides
an alternative approach for comparative genomic sequencing.
In particular, large-insert bacterial artificial chromosome
libraries (BAC libraries) that are currently required to undertake
targeted mapping and sequencing projects are available for many
diverse species, including more than 90 vertebrate (for example,
see
http://bacpac.chori.org). Importantly, a substantial fraction
of genomic libraries represent genomes that have not been subjected
to whole-genome sequencing. Targeted physical mapping and sequencing
can therefore be used to augment existing comparative genomic
data sets from chromosomal regions of interest (
3,
4) and generate
high-quality sequence data in regions of incomplete or low-quality
assemblies generated by whole-genome shotgun sequencing (
5).
The creation of physical maps also provides direct access to
sequenced BAC clones that can be used in a variety of experimental
settings. It is therefore important that efficient methods be
available for undertaking targeted comparative mapping and sequencing
projects.
Targeted physical maps are typically constructed by screening a BAC library with a set of hybridization-based probes that correspond to the chromosomal region of interest. Species-specific ‘overgo’ probes, which are 36–40 bp radioactively labeled double-stranded DNA probes (6), have been shown to be exceptionally useful for screening genomic libraries (7). Overgo design programs include, Overgo Maker, (http://genome.wustl.edu/tools/software/overgo.cgi), which takes as input nonaligned *.fasta formatted files, and OligoSpawn (http://138.23.191.145/) (8), which can design either ‘unique’ or ‘popular’ overgo probes that respectively occur once, or more than once in a provided set of sequences. However, for a species for which a BAC library is available but for which no large-scale DNA sequence resources are available, the design of species-specific overgo probes is often not feasible. Previously, we showed that by considering sequence conservation in the design of overgo probes, ‘universal’ probes could be developed for efficiently screening one or more genomic library (9). Universal overgo probes therefore alleviate the need for species-specific probes and can be used in the construction of physical maps of orthologous regions from multiple species in parallel (9). To make this method more accessible, we systematically applied the universal probe design concept to whole-genome alignments and have disseminated the resulting probe sets for screening mammalian and bird/reptile BAC libraries through a website called Uprobe (http://uprobe.genetics.emory.edu) (10).
Here, we report the creation of three new predesigned whole genome sets of universal probes for screening marsupial, rodent and carnivore genomic libraries, advanced capabilities for searching the predesigned universal probe sets, a tool for the on-demand design of universal overgo probes for screening ape and old world monkey genomic libraries, and finally the capability for the custom design of universal overgo probes from user supplied DNA sequence alignments.
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OVERVIEW OF THE UNIVERSAL OVERGO DESIGN PROCESS
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An overview of the steps taken in the design of universal overgo
probes by the Uprobe website is illustrated in
Figure 1. Briefly,
an entire genome, or target region within a genome, is selected
for probe design. Pairwise or multiple-species whole-genome
alignments are processed with the nsoop_v2 algorithm, which
incorporates the phylogenetic relationship among the species
to identify the most conserved 36-mers suitable for overgo probes
that can be used at a standard hybridization and washing temperature
of 58°C (i.e. 36-mers with a GC content between 44% and
56%) (
9,
10). Potential overgo probes are then compared back
to the genome of origin and/or additional genomes to identify
probes which will likely hybridize to a single locus in the
genome (i.e. are unique), or more than one location (i.e. are
nonunique). A subset of the unique probes that are optimally
spaced along the chromosome for constructing a probe-content
physical map of the chromosomal region of interest is then selected,
and the primer sequences and accompanying information for each
overgo probe is provided to the user.
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PREDESIGNED WHOLE-GENOME PROBE SETS FOR MARSUPIALS, RODENTS AND CARNIVORES
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To expand on the predesigned whole-genome sets of universal
probes for screening mammalian and bird/reptile genomic libraries
available at the Uprobe website, three additional subclades
of mammals, marsupials, carnivores and rodents, were selected
for the development of whole-genome universal probe sets. In
each case, whole-genome alignments were downloaded from the
UCSC Genome Browser (
11) and used as the basis for universal
probe design. The marsupial universal probe set was developed
from the assembled opossum genome (monDom1) in regions conserved
with the human genome (hg17), whereas the rodent and carnivore
probe sets were derived from the mouse (mm5) and dog (canFam1)
genomes, respectively, in regions conserved between the mouse,
rat (rn3), human and dog genomes. Unique probes from the marsupial,
rodent and carnivore probe sets are spaced on average every
29, 9 and 6 kb, respectively (
Table 1) (
10), and with the exception
of the marsupial probe set that had the lowest density of probes,
provide coverage of the genome comparable to that of the previously
described mammalian and bird/reptile probe sets (
Table 1). Note
that like the previously described mammalian and bird/reptile
whole-genome probe sets, the coverage across the genome for
the new probe sets are nonuniform and dependent on a variety
of factors including the density of conserved sequences (data
not shown) (
10). To validate the design properties used to develop
each probe set, samples of 48 representative probes consisting
of 4–8 linked probes from 10 different regions of the
genome were experimentally tested by screening one or more genomic
libraries from the target clade of interest. The resulting probe
success rates, as defined by the percentage of probes that successfully
identified at least two but fewer than 20 clones in each
10
x library, were as follows: marsupial—75% for wallaby (ME_KBa);
rodent—31% for squirrel (VMRC-20) and 83% for deer mouse
(CHORI-233); carnivore—73% for clouded leopard (CHORI-87)
(
Table 1). To assess the combined ability of the clusters of
linked probes to specifically isolate the orthologous genomic
regions of interest in each species, probe-content and restriction-enzyme
fingerprint mapping data were used to select representative
sets of BAC clones for sequencing (
Table 1). Two-thirds of the
sequenced marsupial BAC clones and 100% of rodent and carnivore
sequenced BAC clones mapped to the targeted orthologous region.
The genome coverage, probe success rates and sequence validation
therefore demonstrate that the marsupial, rodent and carnivore
whole-genome predesigned universal probe sets can be successfully
used for targeted physical mapping in each of these clades.
Note that more details regarding the probe design process and
experimental validation of each of these probe sets, including
files describing the specific probes that were tested and the
mapping results, is provided on the Uprobe website (
http://uprobe.genetics.emory.edu/direction.php).
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BATCH AND CROSS-SPECIES OPTIONS FOR UNIVERSAL PROBE RETRIEVAL
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The Uprobe website was originally designed to support a variety
of text-based queries, such as chromosome location, gene name
or GenBank accession number, to identify the genomic region
of interest and the corresponding universal probes that map
to that interval of the genome. However, batch queries were
not supported, and queries needed to be in reference to the
genome from which the probes were designed. To expand the ability
of users to search for universal probes of interest, we implemented
a pair of advanced search options (
http://uprobe.genetics.emory.edu/advanced_search.php)
that specifically are designed to handle batch queries or cross-species
searches. With the batch query tool, probes from multiple loci
can be searched for simultaneously by inputting a list of text
queries, thus eliminating the need to sequentially enter queries
for single loci. The cross-species tool provides the capability
of directing a query not only at the genome from which the probes
were derived, but also one of the comparative genomes used in
the probe design process. For example, a user may want to initiate
a comparative mapping and sequencing effort in marsupials orthologous
to a defined chromosomal region in the human genome. With the
cross-species application it is possible to retrieve probes
from the marsupial probe set based on their syntenic location
in the human genome. In other words, the cross-species application
will translate a user-defined interval in the human genome to
the orthologous position in the marsupial genome, thereby identifying
probes from the chromosomal region of interest. These enhanced
query capabilities, which were in part in response to feedback
from the public, provide useful additions to the search options
available to the Uprobe website.
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ON-DEMAND DESIGN OF UNIVERSAL PROBES FOR APES AND OLD WORLD MONKEYS
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BAC libraries representing 16 different species of nonhuman
primates are now available (
http://bacpac.chori.org) (
12), and
represent a unique and valuable resource for reconstructing
the history of the human genome, as well as for comparative
studies of the sequenced genomes of nonhuman primates used in
biomedical research, like Rhesus macaque (old world monkey)
(
13), and marmoset (new world monkey). In order to facilitate
targeted mapping and sequencing efforts in nonhuman primates,
we developed an on-demand universal probe design tool focused
on these species. Similar to the query interface to the predesigned
universal probe sets, text-based queries of chromosome location,
gene name, accession number or keyword are used to initiate
the probe selection process. Universal probes are then designed
with a set conservation criteria optimized for the clade of
interest and returned to the user via an email. However, unlike
the predesigned probe set application, the on-demand tool designs
probes on-the-fly in response to each query. A major benefit
of the on-demand probe design pipeline is that it formed the
basis of a more flexible probe design tool that could be implemented
in other settings (i.e., the custom probe design tool, which
is described in the next section).
At the current time, the on-demand tool supports the design of universal probes for screening ape and old world monkey genomic libraries. The universal probes for this set of species are derived from the human genome (hg18) conserved sequences identified by using the human, chimpanzee (panTro1) and macaque (rheMac2) whole-genome alignment downloaded from the UCSC Genome Browser. Similar to the cross-species advanced search option described earlier, queries using this tool can be directed at locations in either the human or macaque genome. The estimated average probe spacing for ape and old world monkey universal probes is 1 unique probe every 2 kb, and the experimentally determined success rates for the probe design criteria were: 94% gibbon (CHORI-271), 94% Japanese macaque (CHORI-270), 83% baboon (RPCI-41) and 83% colobus monkey (CHORI-272) (Table 1). Compared to the predesigned universal probe sets, the ape and old world monkey probes are found at a greater frequency in the genome and have a higher minimum probe success rate (Table 1). Both these observations are a consequence of the relatively low divergence among apes and old world monkeys compared to the more evolutionary diverse groups of species targeted previously. In addition, sequencing of representative BAC clones indicated that the combined specificity of the probes was very high and ranged from 86% in the colobus monkey to 100% in gibbon and the other two old world monkeys (Table 1). Thus, this application can provide a highly efficient method of designing probes for targeted physical mapping and sequencing in apes and old world monkeys. Future upgrades to the Uprobe site will include expanding the on-demand tool to other clades of nonhuman primates.
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CUSTOM UNIVERSAL PROBE DESIGN TOOL
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A limitation of our predesigned probe sets and on-demand probe
design tool is that they are not capable of continuously incorporating
the ever expanding list of genome sequence assemblies (
14) in
the probe design process. In addition, while our probe design
efforts have been focused on a select set of vertebrates, the
universal probe design concept could be applied to any set of
species for which comparative sequencing data are available.
We therefore developed a custom universal probe design tool
based on our validated on-demand probe design pipeline that
allows the custom design of universal probes based on user-provided
sequence alignments. Initiated by the upload of an alignment
file to the Uprobe website, the custom universal probe design
tool walks the user through the necessary steps of probe design
illustrated in
Figure 1. Help and example files are provided
for each step of the process on the website. Though primarily
designed to process maf formatted alignment files that can be
directly downloaded for any given region of a reference genome
from the UCSC Genome Browser, the custom probe design tool will
also accommodate probe design from fasta, blastz, clustal, phylip
and lagan formatted files. After going through the full set
of required steps, including selection of a reference sequence
for probe design and comparative species, confirmation of a
phylogenetic tree describing the set of species to be used in
probe design, selection of optimal probe spacing and type of
probe (unique or nonunique), the best set of conserved probes
is returned to the user via email. Note, that unlike the predesigned
probe sets and probes generated from the on-demand tool, no
minimum conservation threshold is implemented in the custom
pipeline. However, an output file is generated that includes
the alignment information and conservation ‘score’
for each overgo that can be postprocessed by the user to identify
sequences conserved at a given threshold. The custom probe design
tool therefore allows the public to apply our proven universal
overgo probe design pipeline for the development of universal
probes from DNA sequence alignments between any given set of
species.
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CONCLUSION
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The Uprobe website, first described in 2005 (
10) has served
as a unique online resource for universal overgo-hybridization
retrieval and design. Since the initial development of Uprobe,
many new genomes have been sequenced, thus greatly expanding
the capability to design universal probes. Here, we have described
the updates to the Uprobe site, including new search options,
as well as capabilities that have exploited these new sequence
resources, i.e. new whole-genome probe sets for screening marsupial,
rodent and carnivore genomic libraries, an on-demand tool for
universal probe design for screening nonhuman primate genomic
libraries, and a custom probe design tool. As such, Uprobe provides
a novel online tool for targeted comparative physical mapping
in vertebrates and other species.
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APPENDIX
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The GenBank accession numbers are as follows: AC217456.1, AC217457.1,
AC217464.1-AC217469.1, AC217471.1, AC217474.1-AC217478.1, AC217481.1,
AC217485.1, AC217592.1-AC217594.1, AC217599.1, AC217600.1, AC217614.1,
AC217621.1, AC217738.1-AC217740.1, AC217745.1-AC217751.1, AC217765.1,
AC217766.1, AC217856.1, AC217857.1, AC217864.1, AC217879.1,
AC218054.1-AC218056.1, AC218064.1, AC218065.1, AC218076.1-AC218078.1,
AC218087.1, AC218890.1, AC218891.1, AC222517.1, AC222544.1,
AC222548.1, AC222549.1, AC225042.1, AC225044.1, AC225053.1,
AC225055.1, AC225056.1, AC225057.1, AC217748.1, AC225272.1-AC225275.1,
AC225257.1-AC225259.1, AC225265.1, AC225266.1, AC225276.1, AC225304.1.
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ACKNOWLEDGEMENTS
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The authors wish to thank the NISC Comparative Sequencing Program
for their testing of the website and the specific contributions
of E. D. Green, R. T. Blakesley, G. G. Bouffard and P. J. Thomas,
and G. K. Tharp for his help debugging the website. This research
was supported in part by the Intramural Research Program of
the National Human Genome Research Institute, National Institutes
of Health. RTS, CBM and JWT were supported by a grant from the
National Institutes of Health (R24RR022239). Funding to pay
the Open Access publication charges for this article was provided
by a grant from the National Center for Research Resources (R24RR022239).
Conflict of interest statement. None declared.
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Footnotes
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The list of GenBank accession numbers are given in the
Appendix.
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