The Human Genome Project has resulted in the elucidation of the genomic sequence of a number of model organisms as well as a reference sequence for the human genome. The utility of these available genomes has been demonstrated by researchers throughout the world, and spurred the desire to obtain additional genomic information from a number of sources. The United States Department of Energy s Joint Genome Institute has undertaken a project to sequence the genome of the amphibian Xenopus (Silurana) tropicalis. The primary goal of the project is to produce a high-quality genome sequence and annotation to meet the needs of the research community. In March of 2002, a number of Xenopus researchers from around the world met at the JGI Production Genomics Facility in Walnut Creek, California to discuss goals and strategies for the project. The project is designed to make use of a whole-genome shotgun approach supplemented with extensive BAC end sequences and shotgun sequence from selected BACs. A high-quality draft genome is desired that will meet minimal criteria for contiguity and long-range linking information. At depths of 6-8X sequence coverage, we expect that a large fraction of features of interest (exons, promoters and regulatory regions) will be covered in large contigs of high sequence quality without gaps. In addition, long-range linking of contigs will be achieved through paired end gap-spanning clones so that contigs are ordered and oriented into large scaffolds with gaps of defined size. These scaffolds typically contain multi megabase-sized regions of the genome. This approach has led to high-quality draft genomes of the pufferfish (Fugu rubripes), Ciona intestinalis and the mouse. Since there will be extensive coverage of large inserts for Xenopus including BAC and Fosmid end sequencing, clones will be readily available for finishing selected regions of the genome.

The US DOE Joint Genome Institute (JGI) mission is to provide the scientific community with high-quality finished genomes. Approximately 300 microbial genomes are currently in the JGI pipeline and to date, 65 have been completed. The objective of the Microbial Finishing laboratory is to process sequencing reactions in order to close physical gaps, sequence gaps, and to increase quality of reads. Since most of the genomes contain complex regions which are difficult to sequence with standard protocols, the lab must use a multitude of techniques specialized for each project. Problematic regions, for example, can be GC-rich or contain hairpin loops, have long homopolymer stretches, can be AT-rich, and/or contain tandem repeats of variable length. Gap closer in such regions is expensive as well as time-consuming, since it requires extensive troubleshooting strategies. Approaches include, optimizing reaction conditions, applying various sequencing chemistries, sequencing the opposite strand, and additional manual editing. For genomes with ? 65% GC content, we use a four step approach to sequence through difficult regions: DMSO, Sequence Finishing Kit (SFK), PCR, and shatter libraries. This strategy has allowed JGI ? s Microbial Genome Finishing Group to complete a number of complex microbial projects, such as, Frankia (~75% GC-rich) and Thermobifida fusca (~68% GC rich).

The purpose of the recently established EST program at DOE-JGI is to produce high quality EST data and full length cDNA sequence. This is used in support of gene-model training, genome annotation, identification of novel transcripts, and for clone resource generation. Since its inception in mid 2003, the program has grown from processing 4 species to 27 species resulting in a total of ~3 million EST sequences. In order to handle this increase in throughput, an EST processing pipeline was developed. The pipeline serves two purposes, to optimize cDNA sequencing by providing the necessary information to determine the quality of the library and to provide a comprehensive content view across cDNA libraries. An overview of the analysis steps and summaries generated by the pipeline will be described. Results combined with publicly available data are used for intra and inter library comparisons. Many of the EST projects cover regions of the phylogenetic tree having few available EST sequences. One organism of particular interest in the JGI collaboration with the Environmental Protection Agency (EPA), is the species Pimephales promelas commonly known as fathead minnow. The aim of the collaboration is to identify novel transcripts to assist in development of expression analysis tools, specifically microarrays. The ability to generate molecular data for Pimephales promelas in response to environmental exposure will aid in toxicity predictions and chemical risk assessments.

The US DOE Joint Genome Institute (JGI) mission is to provide the scientific community with high-quality finished genomes. Approximately 300 microbial genomes are currently in the JGI pipeline and to date, 65 have been completed. The objective of the Microbial Finishing laboratory is to process sequencing reactions in order to close physical gaps, sequence gaps, and to increase quality of reads. Since most of the genomes contain complex regions which are difficult to sequence with standard protocols, the lab must use a multitude of techniques specialized for each project. Problematic regions, for example, can be GC-rich or contain hairpin loops, have long homopolymer stretches, can be AT-rich, and/or contain tandem repeats of variable length. Gap closer in such regions is expensive as well as time-consuming, since it requires extensive troubleshooting strategies. Approaches include, optimizing reaction conditions, applying various sequencing chemistries, sequencing the opposite strand, and additional manual editing. For genomes with !Y 65% GC content, we use a four step approach to sequence through difficult regions: DMSO, Sequence Finishing Kit (SFK), PCR, and shatter libraries. This strategy has allowed JGI!'s Microbial Genome Finishing Group to complete a number of complex microbial projects, such as, Frankia (~75% GC-rich) and Thermobifida fusca (~68% GC rich).

The JGI Annotation Pipeline is routinely used to predict genes in assemblies of Sanger-sequenced eukaryotic genomes. In this study we instead use annotation to assess competing assemblies of a single genome sequenced using alternative technologies. These technologies promise to vastly lower the cost and thus expand the output of nucleotide sequencing compared to the traditional Sanger method. However, the new technologies also bring new challenges such as short reads and new kinds of sequence errors. At JGI we are exploring ways to incorporate 454 pyrosequencing into the standard JGI genome workflow. One way to exploit the respective strengths of 454 and Sanger sequence may be to derive 'hybrid' assemblies from both. To rapidly and consistently assess hybrid, 454-only (20X), and Sanger-only (4X) assemblies of the genome of the ubiquitous plant pathogen Phytophthora capsici, we developed a short version of our standard JGI Annotation Pipeline. This 'minipipe' predicts genes, and than examines 1) internal stop codons and 2) truncated homologs of genes of Sanger-only (8X) Phytophthora sp. Both metrics serve as surrogates for frameshifts resulting from sequencing errors or polymorphism. Our results suggest that while the rate of 454-related frameshifting is high, the hybrid assembly approach holds promise as a low-cost way of estimating the number of genes in a genome. We conclude that annotation is a quick and simple way to assess the quality of novel assemblies