Proposal for sequencing the Xenopus tropicalis genome.
Collaborators to date: Paul Richardson, JGI, Walnut Creek CA, Dan Rokhsar, UC.
Berkeley and JGI, Richard Harland, UC Berkeley, Rob Grainger, University of Virginia,
Bruce Blumberg, UC Irvine.
Preface to Xenopus White Paper.
This paper is being submitted after the recent announcement that the Joint Genome
Institute is planning an extensive effort to sequence the Xenopus tropicalis genome. As
discussed below, the current plan from JGI is for 6X coverage of the genome using a
shotgun sequencing strategy. This paper is being submitted because in addition to the JGI
commitment, it would be extremely valuable to have complementary support from NIH: to
increase the overall extent of coverage, for BAC clone sequencing, and for some degree of
finishing. Additional support for annotation would also be extremely valuable. A
mechanism for linking data from the sequencing project to the existing Xenopus database,
and funds for expanding that database would be of great value to the Xenopus community as
well.
A meeting at JGI in mid-March will plan out the sequencing strategy and discuss the
process for annotation in more detail, allowing a more thorough description of anticipated
needs to be then forwarded to NIH. Because of the high level of NIH support for Xenopus
researchers, both for individuals and for the community, and because of the importance of
NIH support of this endeavor in the future, as well as a desire to have input from NIH experts
on genome projects, we felt that it was important to submit this paper in the early stages of
the genome sequencing process.
Scientific community's depth of interest in having the sequence of the particular organism,
and should describe any process, if there has been one, by which that research
community has come to consensus on the request being made.
The community of Xenopus cell and developmental biologists has been overtaken by techniques
and thinking in the field of genomics. Discussion on the resources that would be needed for
effective exploitation of Xenopus in modern biology culminated in a report in March 2000
(http://www.nih.gov/science/models/Xenopus/reports/Xenopus_report.pdf). Such resources
include the generation of both EST and full-length cDNA sequences, microarrays, databases,
and genetic mapping tools. However, the revolution in sequencing technology and reduction
in cost, as well as recent progress in the development of genetic tools, has now led to a new
priority, namely to sequence the genome of Xenopus tropicalis. As described in detail
below, Xenopus tropicalis is a close relative of Xenopus laevis, sharing the many advantages
of work on the latter species, and adding to it a diploid genome and short life cycle that make
it especially attractive for genetic and genomic studies. The rationale, and discussion of the
commitment of the Joint Genome Institute, Walnut Creek CA is presented below.
The JGI is organizing a Xenopus workshop that will take place in March 2002 in Walnut
Creek to discuss aspects of the genome project with leading experts in the field. Steve Klein
and other representatives of NIH will be present to determine how best to utilize available
resources to complete the project. Teams will be set up to coordinate activities and ensure
that communication between groups is kept open. This white paper will be made available
for comment through the Harland lab web site and the Xenopus tropicalis web site
(http://minerva.acc.Virginia.EDU/~develbio/trop/), and we will request links or postings to
be made through the NIH web site (http://www.nih.gov/science/models/nmm/appb6.html)
and Xenbase (http://xenbase.org).
A. Specific biological rationales for the utility of new sequence data
1. Improving human health. How will the genomic sequence of an organism inform our
understanding of human health and disease? What, if any, is the relevance to the
development of innovative and improved methods of diagnosis, treatment or prevention?
2. Informing human biology. How will the genomic sequence of a particular organism lead
to a better understanding of biological function in the human?
Xenopus is a unique resource for two critical areas in vertebrate biology: early embryonic
development and cell biology. In the former, Xenopus laevis has led the way in elucidating
mechanisms of early fate decisions, patterning of the basic vertebrate body plan, and early
organogenesis. The powerful methods of experimental embryology combined with modern
molecular biology have led to the first understanding of the mechanisms of formation of the
vertebrate germ layers, and patterning of the vertebrate axis, including the first understanding
of the induction and patterning of the vertebrate nervous system. Contributions in cell
biology and biochemistry include seminal work on chromosome replication, chromatin and
nuclear assembly, control of the cell cycle, in vitro reconstruction of cytoskeletal dynamics,
and signaling pathways. In fact, Xenopus has become a major vertebrate model for the
cellular and developmental biology research that is supported by most of the Institutes of the
NIH. Because so many of the processes that have been pioneered in studies in Xenopus as
conserved in all vertebrates, sequencing of the X. tropicalis genome will contribute to more
rapid and profound advances in the understanding of vertebrate biology in general, and by
inference, of human biology and human disease.
It should be emphasized that the classical advantages of Xenopus for understanding cell
and developmental phenomena that are deeply relevant to understanding human biology and
disease have been further amplified by recent advances in the system. Systematic analysis
of the expressed genome, efficient transgenesis, the ability to identify mutations, the
development of expression cloning strategies coupled to study of protein-protein interactions,
and other advances discussed below, have added important new dimensions to the system.
With the addition of a genomic sequence would one could argue that Xenopus would provide
the most uniform and integrated resource for utilizing genome-wide data in any vertebrate.
3. Informing the human sequence. How will the genomic sequence of a particular organism
lead to a better description of the functions of specific sequence features of the human
genome?
4. Providing a better connection between the sequences of non-human organisms and the
human sequence. How will the genomic sequence of a particular organism increase our
ability to identify orthologs in the sequences of well-studied model organisms and how will
that deepen our understanding of the human sequence?
Items 3. and 4 are met by the comparative aspects of sequencing the Xenopus tropicalis
genome. The comparative approach has already shown itself to be valuable in comparisons
of mouse and human sequences. After comparisons of these mammals, the next relatives that
are being sequenced are the bony fishes, Fugu, and the zebrafish Danio rerio. The
amphibians occupy a key phylogenetic position between these mammalian and fish genomes.
Xenopus tropicalis, a close relative of X. laevis, is well-placed, not only to complement
studies of Xenopus laevis, but to extend research into new areas. Two aspects are obvious:
First, Xenopus tropicalis offers one of the smallest genomes among the amphibians (which
number more than 4,500 species). It is the only Xenopus species that is diploid, greatly
simplifying genetic studies. When combined with the mass of research on Xenopus laevis,
Xenopus tropicalis is in a unique position to make fundamental contributions to comparative
and functional genomics of vertebrate animals.
As comparative genomics develops, the Xenopus tropicalis genome will allow insights
into the similarities in development and physiology of the vertebrates, as well as the
important differences in the use of regulatory DNA sequences. For example, amphibians
have a limb skeleton that clearly resembles that of the mammals, but is radically different
from the fin of the fish. Although similar to the mammalian limb, the Xenopus limb is
different in the numbers of digits, and fusion of skeletal elements. We expect that these
similarities and differences will ultimately be understood by a comparative analysis, and lead
to a deep understanding of developmental mechanisms. When combined with the ease of
analysis of regulatory information in transgenic animals (see below), we expect many new
insights and principles to emerge from study of Xenopus tropicalis.
5. Expanding our understanding of basic biological processes relevant to human health,
e.g. developmental biology, neurobiology.
See response to 1 and 2 above. Xenopus and the amphibians have already shown their
importance in understanding basic cell biology, and developmental biology. Having the
sequence available will facilitate the discovery of new functions for genes that have been
identified only as open reading frames, and will allow the ready isolation of Xenopus
homologs of genes that may have been discovered in other species. For gene discovery,
many current approaches use biochemical isolation of complexes, followed by mass
spectrometric identification of peptides, which are used to infer the complete coding regions.
The lack of comprehensive sequence information is already a bottleneck for cell biologists
who use Xenopus egg extracts to study events of the cell cycle.
6. Providing additional surrogate systems for human experimentation, e.g. new disease
models, improved opportunities for drug testing, or other medical procedures, such as
transplantation.
Frogs are currently being developed for use as environmental, teratogen and toxin
indicators. The FETAX assay (Frog Embryo Teratogenesis Assay—Xenopus)
(http://iccvam.niehs.nih.gov/methods/fetax.htm) is important in toxicological and
environmental research. Toxicogenomics (http://www.niehs.nih.gov/nct/home.htm) is a
burgeoning field and the availability of EST and genomic sequences from Xenopus will be
invaluable in developing microarrays and other sensitive high throughput tools. These will be
important in developing a comprehensive list of the genes that are sensitive to environmental
insult.
While the technology for making transgenic lines is still new, investigators are now using
Xenopus to identify genes that control organogenesis, since one can use this technology to
make transgenic reporter constructs to mark key events in specific tissues and to target genes,
and modified gene constructs, to later developmental stages. The ability to make such
transgenic lines coupled with the ability to perform the tissue interaction studies for which
Xenopus is also so well suited, will give the Xenopus system an unparalleled set of tools for
understanding organ formation. Understanding the principles by which various organ
systems form remains one of the major challenges for modern developmental biologists, and
such an understanding will be vital for resolving how organ systems fail during the course of
many diseases and during aging.
7. Facilitating the ability to do experiments, e.g "direct" genetics or positional mapping, in
additional organisms.
Certainly a great value of genome projects in Xenopus tropicalis would stems from its
connection with genetic studies. While genetics in Xenopus tropicalis is a relatively new
endeavor, the system is well suited to genetics, like zebrafish, and progress is rapid.
Mutations are being identified now in several laboratories (over a dozen have been identified
in the Grainger laboratory), either in wild-caught animals, or by using chemical mutagenesis
and gene-trapping strategies. A recent RFA from NIH on Xenopus tropicalis genetics has
helped to spawn great interest in developing and characterizing mutations in this system.
The Xenopus tropicalis system has several advantages, some noted earlier, that will make
it a superb genetic system: rapid lifecycle, external development and large numbers of
gametes are commonly described features of this system. The ability to readily make
gynogenetic animals, permits rapid and efficient screening of recessive mutations (by
obviating one generation of screening by eliminating the need to cross heterozygotes to
reveal recessive phenoytpes). A number of mutations have been already identified by this
method. The ability to readily transplant germ cells in embryos means that lethal mutations
can be carried in wildtype hosts, providing a unique method for easily preparing homozygous
embryos. More generally, the extraordinary ability of tissue healing in Xenopus will permit
one to make genetic chimeras by transplanting mutant tissue into a wildtype background.
The inability to perform this kind of analysis in the mouse, and the limited ability to do so in
zebrafish give the Xenopus system a unique place in genetic studies.
The rapid generation of mutations in an animal that permits these mutations to be easily
characterized (for example by making genetic chimeras) will be an important complement to
a genome sequencing effort. The preparation of an RH panel and extensive mapping of
polymorphisms between two genetically divergent inbred strains, now underway, will lead to
a genetic map that can be used, together with genomic sequence information, to provide a
powerful set of tools for cloning mutated genes. The use of the striking lampbrush
chromosomes in Xenopus oocytes affords another mapping tool not available in mammalian
systems.
One of the most important uses for the genomic information from the DoE perspective
will be to investigate gene regulatory pathways, particularly those involved in DNA repair.
From the NIH point of view there is a broader interest in investigations of developmental
mechanisms. Comparisons between upstream regulatory regions between species may lead
to identification of transcription factors that determine specific patterns of development.
Comparisons of the genome sequence of X. tropicalis with those of the mammals will focus
interest on deeply conserved regulatory motifs and open reading frames. As the
sophistication of bioinformatics techniques improves, we expect that the additional sequence
from an amphibian will make predictions of function in the mammals considerably more
robust. Such conserved features can then be assayed in an experimental system. This work
will build from current functional genomics studies being carried out at the JGI on Ciona
intestinalis, and the resources of sequence information from other organisms in public
databases. Predictions on conserved modes of regulation will be then tested in transgenic
animals.
8. Expanding our understanding of evolutionary processes (biological innovation,
selection) in general, and human evolution in particular.
As referred to above, the amphibians are key in understanding the radiation of the
vertebrates. The DNA sequence will provide an extraordinary resource for understanding
vertebrate evolution. Ultimately, the development of the characteristic frog skeleton, with its
abbreviated spinal column, its different skull and limb morphology, will be understood at the
level of cis-regulatory regions that direct specific patterns of expression of peptide growth
factors, and their antagonists, as well as the array of transcriptional regulators that provide
the ability of the cell to respond to external signals. Xenopus lends itself to comprehensive
functional genomics, whether it is the complete description of how transcription factors are
expressed in the developing embryo, to the comprehensive analysis of transcriptional
regulation by these factors. Transgenic experiments will be combined with loss of function
experiments, mediated by such new tools as morpholino oligonucleotides, to show not only
what regions in DNAs are important in directing expression, but also in proving that
particular trans-acting factors are required to mediate effects through those binding sites. By
understanding the development of this vertebrate in particular with functional genomics,
considerable information will have been gained about what builds an organism, and by direct
comparison with the regulatory DNA and proteins of the mammals, we will have gained a
substantial insight into how human development is controlled, and how the regulatory
mechanisms have changed over evolutionary time.
Human populations have substantial impact on the environment. Frogs and tadpoles
absorb chemicals from air and water through their thin porous skin. For this reason, they are
good indicators of environmental damage caused by pollution. Frogs and other amphibians
have been referred to as the canary in the coal mine, and may provide and early warning to
humans of environmental degradation or pending disaster. Surveys of local frog populations
have been used to monitor water pollution around mining and industrial sites. The numbers
of amphibians and frogs have been declining worldwide for the past several decades.
Possible reasons for this decline include loss of habitat, pesticide pollution, parasitic
infestations, and increased UV light exposure due to the decrease in the ozone layer. Having
a better understanding of the frog genome through this project will give greater insight and
allow for a more comprehensive analysis of the crucial factors in environmental damage and
degradation.
Strategic Issues
1. The demand for the new sequence data. What is the size of the research community that
will use it? What is the community's enthusiasm for having the sequence? Will the new
sequence data stimulate the expansion of the research community?
NIH currently funds 402 research grants utilizing Xenopus with a total award amount of
$86,289,711. In addition, NSF funds an additional 13 awards in the amount of $2,000,000.
The current mailing list for researchers interested in Xenopus research numbers 328
researchers worldwide. A search of Xenopus in the pubMed database revealed citations for
over 25,000 papers. There is a Xenopus database (http://www.xenbase.org) available that
contains an important compendium of useful data, including contact information for over 200
researchers in the field.
The availability of the sequence will stimulate research on comparisons of genomes,
since the Xenopus tropicalis genome sequence will fill the gap between the mammals and the
fish. The Zebrafish genome has shown surprising synteny with the human sequence. We
therefore expect the Xenopus sequence to show substantial synteny, and will be exploited to
understand conserved features of the vertebrate genes and cis-regulatory sequences. Since
Xenopus has become a facile organism for transgenic studies, we expect that there will be a
substantial increase in the number of researchers exploiting Xenopus to carry out detailed
analysis of gene regulation.
The development of genetic analysis will allow a quantum leap forward in the potential
of Xenopus. When combined with available genome sequence information, this will provide
a substantial impetus to expand the community that exploits the amphibian. As a potential
genetic system X. tropicalis presents very important advantages. When compared to Xenopus
laevis it develops to sexual maturity in 1/3 the time, has 1/2 the genome size (and is believed
to be diploid), and requires 1/5 the housing space. It nonetheless shares the embryological
advantages of Xenopus laevis and gene sequences are sufficiently similar that probes crossreact between the two species. Haploid and gynogenetic diploid embryos are easy to
generate; this greatly reduces the time (and space) needed to make inbred lines and to
perform genetic screens when compared, for example, to the resources needed for mouse
genetics. As a vertebrate genetic model, this system offers significant advantages over the
mouse system, which costs far more to house and maintain, and has raised more controversy
as a model because it is a mammal. The evolutionary conservation of developmental
processes revealed in recent years among all vertebrates gives us confidence that information
revealed from studies of lower vertebrates will, in large part, apply to mammalian systems.
The utility of a genetic approach in frogs over mice is highlighted in other ways as well.
Simply having the large numbers of eggs in a typical Xenopus spawning is of great value. Of
course, ability to manipulate the frog embryo is significantly better; an advantage that is
underscored when one considers the possibility of making genetic chimeras. In frogs it
should be possible to make chimeras of mutant and wild type tissue to be able to very readily
study the effects of gene manipulation on particular developmental events by obviating early,
more general lethal effects that have diminished the ability to interpret many mouse
knockouts. The urgent need for developing a genetic component to Xenopus research now
emerges when one considers the implications of recent breakthroughs arising from this
system. For example, findings on signal transduction through the Wnt and BMP pathways
have resulted from analysis in X. laevis. However, as we try to integrate the many
components of these pathways the ability of reagents traditionally used in this system (e.g.
antisense and dominant negatives) are very limited when compared to the prospect of having
mutations in these genes for epistasis studies. The same principle applies to tissue and organ
formation, where, for example, in the eye, brain and heart, many regulatory genes have been
identified but the subtleties of gene interaction remain poorly understood because there are
no developed genetic tools.
To take full advantage of the Xenopus system the genetic dimension must be developed
together with the genomic sequence information: the genomics will greatly enhance the
ability of, and enthusiasm for, tackling genetic manipulations. For example, as noted earlier,
the availability of a genomic sequence, coupled with ongoing work to make RH panels and
an extensive effort to map polymorphisms in genetically divergent strains, would provide the
resources needed for identifying genes affected in mutations generated by mutagens like
ENU.
2. The suitability of the organism for experimentation. What are the basic properties of the
organism that affect its ability to be studied in the laboratory (e.g. availability, ability to be
cultured and propagated in the laboratory, generation time)? Are mutants available with
defined phenotypes? How will the new sequence data enhance the experimental use of the
organism? What other genomic resources and technologies (e.g. gene transfer, ability to go
from molecule to mutation) are available that will allow the new sequence information to
be effectively used?
Xenopus tropicalis, and its relative Xenopus laevis are readily maintained in the
laboratory, and are commercially available. Unlike many amphibians, they are not seasonal
egg-layers, and can be stimulated to ovulate several hundred eggs by injection of mammalian
chorionic gonadotropin. The embryos develop externally and are readily manipulated. The
embryological techniques and molecular assays which have been described for X. laevis are
readily applied to X. tropicalis, but may be supported by multigeneration genetic analyses.
The diploid genome of X. tropicalis will facilitate identification of authentic gene
regulatory regions, rather than pseudogenes which are frequently recovered in the
pseudotetraploid X. laevis. These cis-regulatory regions can be analyzed either by
transgenesis in Xenopus laevis or X. tropicalis.
Xenopus tropicalis has a generation time of a few months, which when combined with its
likely diploid state, make it a suitable organism for genetic research (see also Section A7).
Pilot mutageneses are in progress in both the U.S. and in Europe. At present over a dozen
mutants have been identified in the Grainger laboratory, which include phenotypes associated
with heart formation, left-right asymmetry in gut and heart tissues, and forebrain, eye and ear
development. With the availability of the genome sequence, and the development of
mapping techniques noted earlier (particularly those that exploit the large numbers of
meioses that can be recovered from Xenopus), the sequence will be exploited to identify
lesions in mutated genes. Indeed the rapid pace of genomic advances with this organism
have stimulated interest in its use for forward genetic analysis. The sequence information
will be combined with the BAC resources, and EST libraries for rapid identification of gene
regulatory regions.
Resources available to complement the sequence.
NIH has funded or is considering funding several areas of research related to Xenopus
genomics.
Prof. Rob Grainger is leading an effort to produce a large insert BAC library that will be
for mapping and end sequencing. That library is now under construction. A second smaller
insert BAC library is currently being constructed at the Institute for Systems Biology in
Seattle. These two libraries will be valuable resources that will be freely available to the
research community.
Xenopus laevis and tropicalis EST projects are currently underway with 198,119 ESTs
available for Xenopus laevis and 43,464 ESTs from Xenopus tropicalis. The limiting step for
X. tropicalis has been the availability of high quality cDNA libraries. Bruce Blumberg at the
University of California, Irvine has recently received a grant from NCRR to generate
eighteen normalized, full-length enriched cDNA libraries from Xenopus tropicalis over the
next three years. These will be made available to the community at large without restriction
and will serve as an important genomic resource for functional studies and working out the
details of transcription units from the BACs and genomic sequence when it becomes
available. Exploring another dimension, the cDNA libraries alluded to above are already
being used for automated, large scale, protein-protein interaction screening based on IVEC
(in vitro expression cloning) technology, and should be a vital link in allowing researchers to
go from gene identification to tests of protein function in short order.
Ali Hemmati-Brivanlou at Rockefeller University has been funded to make cDNA
microarray chips available to the community. Peter Vize (University of Calgary) is funded to
maintain and expand the Xenopus database. Funding opportunities also exist to conduct pilot
genetic studies, prepare radiation hybrid maps and develop training and stock centers.
Peter Vize, at the University of Calgary, maintains the Xenopus database
(http://xenbase.org) and has bioinformatics resources that will permit him to begin to
integrate the existing database with information generated by EST projects and a genomic
sequence effort.
3. The rationale for the complete sequence of the organism. Why would the complete
sequence be more useful than the sequences of specific regions, or only the coding
sequences, or only ESTs? Are there alternative ways to get the necessary information?
Having the complete sequence for Xenopus tropicalis will be very informative for protein
coding regions as well as for identifying potential regulatory regions. Saturating EST
coverage would be useful, but would not tell the whole story, as has become clear from the
human, Drosophila, and C. elegans genome projects. One distinct advantage of Xenopus for
genomic studies lies in ready transgenesis for analysis of cis-regulatory DNA and this
requires genomic sequence. It should be emphasized here that it is feasible for one person to
generate 100's of transgenic embryos in Xenopus in a single day, making it arguably the most
efficient vertebrate for performing promoter analyses.
In addition, the rationale for comparative genomics, where the sequence will be exploited
to understand relations of regulatory regions and coding regions between different animals,
requires comprehensive sequence analysis. It would be very advantageous to have the full
genomic sequence for whole genome comparisons between human, mouse, frog, fugu,
zebrafish, ciona, fly and the worm. As stated above, Xenopus occupies a key phylogenetic
position in this list. Computational tools being developed such as the VISTA programs from
Dr. Eddy Rubin's lab will allow detailed comparisons of large regions of the genome that
may shed light on conserved coding and non-coding regulatory sites. The additional
information obtained from the whole genome sequence is critical, particularly for identifying
key regulatory events. This is especially important for taking advantage of the large body of
work on early development that has been performed in Xenopus to date, and will greatly aid
and direct future experiments. With the availability of complete sequence information we
anticipate that X. tropicalis will become an exceptional organism, perhaps even the organism
of choice, to understand the functioning of the vertebrate genome.
4. The cost of sequencing the genome and the state of readiness of the organism's DNA for
sequencing. What is the size of the genome? What quality of sequence product is needed
(finished sequence? draft? full shotgun?)? What sequencing strategy will be used? Is
suitable DNA readily available?
Cost: Since the bulk of the costs will be borne by the DOE budget at JGI, the NIH is
being asked to support only a part of the total costs (see below-other sources of funding
available)
State of readiness and suitability of DNA.
The Nigerian strain of X. tropicalis is now in the seventh generation of inbreeding. AFLP
analysis of polymorphisms in these strains and their crosses, as well as in wild-caught
animals, indicate that the Nigerian line started with fewer polymorphisms than the other
strain in use (from Ivory Coast). Seven generations of brother-sister inbreeding is often used
as the standard for defining an “inbred” line in many organisms. Sixth generation animals
are available now and seventh generation animals will be mature within two months. These
inbred X. tropicalis animals will serve as an excellent source of DNA. Like all amphibians,
Xenopus has nucleated red blood cells. These metabolically quiescent cells, with compacted
DNA, and absence of nucleases, are the ideal source for preparation of genomic DNA of the
highest quality. DNA will be prepared from a single individual for shotgun sequencing. The
limited polymorphism will contribute substantially to the ease of assembly of the finished
sequence.
Size of genome estimated at 1.7 GB.
Strategy
JGI will likely sequence from several libraries containing insert sizes of varying length.
For instance, the majority of reads will come from a 4-5kb plasmid library, constructed from
a single inbred frog.
We will also construct 10kb plasmid libraries as well as larger insert phage libraries. A
40kb Fosmid library will also be constructed and a portion of that will be end -sequenced.
Having sequence from a number of different insert sized libraries aids the assembly by
spanning long repeats and other specific sequences unique to each genome. Each of these
libraries ideally will be constructed from a single DNA source.
This is adequate to provide a usable assembly, but is not quite optimal. Additional
funding to bring the total to 8x or above would certainly improve the assembly. In terms of
having a more complete assembly with larger scaffolds containing whole genes, it would be
very good to have longer range map information. If a good physical map could be obtained
during the time-frame of the sequencing project, that information could be used to pick
BACs from which small insert libraries would give some coverage to overlay on the whole
genome shotgun reads. This may be very useful if there is good mapping information.
Some portion of the available BAC libraries will be end-sequenced. These reads are
placed on the assembly and if there is enough coverage, can be used to order and orient large
scaffolds. This can be very useful in creating larger scaffolds. Ideally, we would be looking
for end sequences on enough BACs to provide appoximately 8-12X clone coverage across
the genome. BACs that can be placed at specific locations in the assembly can become
resources for finishing. While finishing the entire genome to Bermuda quality may not be
called for here, if we localize BACs on the assembly (using end sequence or map
information), specific BACs could be pulled and used for finishing. EST sequences are
added in the final stages of assembly to help annotate the genome. These are often a rich
source of information by confirming predicted genes and providing tissue specific expression
information. SAGE sequencing may also provide significant gene expression information
which can also be useful for annotation.
5. Are there other (partial) sources of funding available or being sought for this sequencing
project? All proposals of new organisms for genomic sequencing by the NHGRI-supported
sequencing centers must address both sets of issues. If one or more of the issues listed above
is not applicable to the specific request that should be stated clearly rather than not
mentioned.
JGI is committed to initiating sequencing of the Xenopus tropicalis genome starting in
2002. At this time, DOE intends to fund about 6X worth of shotgun sequencing. . The
current costs are estimated to be on the order of $5-6 Million per 1X of whole genome
coverage. The schedule and strategy will be discussed at the upcoming workshop at the JGI
in March. We anticipate beginning the whole genome sequence probably in the late summer
of 2002.