Xenopus tropicalis Genome Sequence

The JGI is sequencing the genome of X. tropicalis with the goal of producing a high-quality draft assembly. The DNA used for construction of whole genome shotgun libraries was isolated from two inbred individuals from the Nigerian strain of X. tropicalis (F6 and F7). The strategy involves end sequencing of approx 4x sequence coverage of a 3-kb insert plasmid library, approx 4x sequence coverage of an 8-kb insert plasmid library, and paired end sequences representing about 15x clone coverage of approx 35-kb insert fosmids. Additional end sequence generated at JGI will be combined with BAC end sequence and mapping information produced at the Washington University Genome Sequencing Center to produce a final assembly. The complete mitochondrial genome has also been assembled and annotated (http:// genome.jgi-psf.org/xenopus0/X.tropicalisMtGenome.gb).

Four preliminary assemblies have been produced to date using JAZZ, the JGI genome assembler. The most recent assembly (version 4) used paired end sequencing reads at a coverage of 7.65x. The version 4 assembly is available for BLAST analysis and downloading at http://genome.jgi-psf.org/Xentr3/Xentr3.home.html. After trimming reads for vector and quality, 22.5 million reads assembled into 19,759 scaffolds totaling 1.51 Gb of genomic sequence, in agreement with previous estimates of the genome size. Approximately half of the genome is contained in 272 scaffolds, all at least 1.56 Mb long. This assembly and preliminary automated annotation are displayed through the JGI genome browser.

A preliminary set of 33,749 gene models was created using the JGI annotation pipeline and is composed of known X. tropicalis genes mapped onto the genomic sequence, homology-based, and ab initio gene models. Additional support for the predicted gene models was provided from available ESTs and cDNAs from X. tropicalis and X. laevis.

This assembly will be the basis for comprehensive annotation and analysis. The larger research community will assist with the manual examination of the automated annotation. A publication on the global analysis of the genome will be submitted soon.

2.8. Microarrays

Microarrays are important tools for gene discovery and for investigating gene function. Several groups are developing Xenopus microarrays. For example, the Affymetrix company has made an X. laevis oligonucleotide microarray that represents over 14,400 transcripts. The transcripts were selected by a group of Xenopus experts and bioinformatics experts from NCBI's UniGene project (process described at http:// www.xenbase.org/genomics/microarrays/Xenbase_affy_upd_v5.html).

The chip includes about 10,400 EST-based gene clusters and about 3500 genes that were well characterized at design time (June 2003). Each transcript is represented by 25mer oligonucleotide probes in a set of 16 pairs. Each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide. A complete list of the transcripts and the sequence of each probe is available at www.affymetrix.com.

The initial lot of about 90 chips was distributed among nine Xenopus labs, including the labs of those who helped design the chip. Preliminary examinations showed that the chips have a good dynamic range and a high degree of reproducibility. The chips are sold by Affymetrix (www.affymetrix.com). Data from this chip are included in NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GPL1318).

2.9. Mutagenesis and Phenotyping

One of the benefits of X. tropicalis is that its diploid genome and shorter generation time should make it amenable to genetic analyses. The addition of genetics to Xenopusâ– ' other advantages could benefit many types of studies. Several projects have begun to examine the potential of X. tropicalis as a genetic model. They are devising and optimizing strategies for mutation induction using a variety of mutagenesis strategies, including chemical mutagenesis, insertional mutagenesis, and y-ray mutagenesis. They are also devising strategies to screen for altered phenotypes at different stages of development and in different organ systems.

So far, these projects have identified many mutagenized animals with interesting phenotypes. They can be divided into animals with alterations in specific organ systems and developmental events, including the cardiovascular system, the digestive system, the ear, and the eye and in axial patterning. For examples, see the Stemple/ Zimmerman project at http://www.sanger.ac.uk/Teams/Team31/phenotypes.shtml. These putative mutants are back-crossed and sib-crossed to determine whether their phenotypic alterations are in fact heritable. So far, several of the interesting pheno-types have been determined to result from heritable mutations.

One of the goals of these projects is to serve as a community resource by disseminating the mutagenesis protocols and screening strategies that they devise and by making their mutant and transgenic strains available to the community. Each project has a Web site that describes their goals, strategies, and protocols and provides a description of the putative mutants and of the mutant strains. They also explain how investigators can obtain mutant strains. Links to their Web sites may be found at the NIH Xenopus Initiative's Web site, http://www.nih.goV/science/models/Xenopus/resources/I.html.

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