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Halorubrum lacusprofundi is an extreme halophile within the archaeal phylum Euryarchaeota. The type strain ACAM 34 was isolated from Deep Lake, Antarctica. H. lacusprofundi is of phylogenetic interest because it is distantly related to the haloarchaea that have previously been sequenced. It is also of interest because of its psychrotolerance. We report here the complete genome sequence of H. lacusprofundi type strain ACAM 34 and its annotation. This genome is part of a 2006 Joint Genome Institute Community Sequencing Program project to sequence genomes of diverse Archaea.

Halorubrum lacusprofundi is an extreme halophile within the archaeal phylum Euryarchaeota. The type strain ACAM 34 was isolated from Deep Lake, Antarctica. H. lacusprofundi is of phylogenetic interest because it is distantly related to the haloarchaea that have previously been sequenced. It is also of interest because of its psychrotolerance. We report here the complete genome sequence of H. lacusprofundi type strain ACAM 34 and its annotation. This genome is part of a 2006 Joint Genome Institute Community Sequencing Program project to sequence genomes of diverse Archaea.

Chromohalobacter salexigens is one of nine currently known species of the genus Chromohalobacter in the family Halomonadaceae. It is the most halotolerant of the so-called ‘moderately halophilic bacteria’ currently known and, due to its strong euryhaline phenotype, it is an established model organism for prokaryotic osmoadaptation. C. salexigens strain 1H11T and Halomonas elongata are the first and the second members of the family Halomonadaceae with a completely sequenced genome. The 3,696,649 bp long chromosome with a total of 3,319 protein-coding and 93 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2004

As a user facility, The US Department of Energy?s Joint Genome Institute, in collaboration with scientists around the world, are able to generate DNA sequences for a diversity of organisms. Often times, the amount of DNA provided for library construction is limited. It is important to develop a protocol to minimize the amount of DNA required for library construction. In an attempt to test the minimum amount of DNA necessary for library construction, we decided to use two approaches to clone chloroplast DNA. The first approach was AMPure bead purification, using solid-phase paramagnetic bead technology to purify DNA fragments from contaminates and enzymes, with minimal loss of DNA. DNA samples used for this approach ranged from 100ng to 1ug. The second approach used was, Multiple Displacement Amplification (MDA), requiring as little as 10pg of DNA template to amplify up to micrograms of DNA. The AMPure bead purified and MDA samples were cloned into pUC18 to determine the quality of the libraries. Several metrics were used to measure the quality of the libraries which include: cloning efficiency, chimera rate, and coverage biases amongst the two techniques. We will present the data generated and the pros and cons from these two different approaches.

As Sanger sequencing is being replaced by higher throughput and lower cost of next generation sequencing, finishing microbial genomes will face two major challenges. First, the technology will need to be fast enough to handle many more drafted genomes. Second, it will have to incorporate a clone-free approach to fill gaps. We have been testing a method that utilizes a universal "bubble-tag" to perform primer walking and gap closure in a clone-free condition. The ?bubble-tag? method was first described by Doug Smith (PCR Methods Appl. 2: 21-27, 1992) to amplify and sequence lambda DNA. There is no evidence however, that this approach will work for the more complex microbial genome. Here we describe the experimentation of this approach in primer walking of the E. coli genome. Genomic DNA was sheared, blunt-end repaired, or digested with frequent cutters, and ligated to bubble adaptors. Site specific primers were used together with the universal bubble primer to amplify and sequence the regions of interest. Different primer concentration was tested in order to generate long amplified templates. This approach enables primer walking and gap filling in a clone-free draft sequencing process. More importantly, the uniformity of this approach is amenable for an automated finishing process.

The US Department of Energy's Joint Genome Institute (JGI) is a user facility for high-throughput sequencing that has sequenced a large number of the nearly 1000 microbial genomes completed or nearing completion. This sequence has enabled us to understand the roles of microorganisms in important processes such as bioremediation, energy production, global nutrient lifecycles, pathogenesis, and the diversity of life. However, the currently available genome sequences are heavily biased based upon their phylogenetic distribution when compared to the microbial genome diversity known to exist. To overcome this bias, the JGI has initiated a project known as the Genomic Encyclopedia of Bacteria and Archaea (GEBA). The long-term goal of the GEBA project would be to generate reference genomes for every major and minor group of bacteria and archaea. To test the feasibility of the project, JGI has launched a pilot project in collaboration with DSMZ to sequence 100 bacterial and archaeal genomes based on their phylogenetic positions in the tree of life. The pilot project could expand to represent 5,000 genomes. Selection of the microbes was based upon analysis of the rRNA tree of life as well as consultation with a scientific advisory board. Partaking in a project so large will present unique obstacles. The JGI will use this pilot study to understand and establish new processes to deal with the potential number of samples this long-term project might include. GEBA s pilot project progress, challenges, and developments will be presented.

The basidiomycete Melampsora larici-populina is responsible for the leaf rust disease in Populus species. Urediniospore germlings of this obligate biotrophic fungus usually penetrate the host plant through stomatal openings, differentiates a series of infection structures in the intercellular space, and exhibits highly localized penetration of the host cell wall to establish a haustorium. Hyphae then proliferate in the leaf parenchyma and produce golden pustules filled with masses of urediniospores on the lower leaf surfaces. M. larici-populina causes severe economic losses in European poplar plantations and has recently been detected in Canada. Selection for resistance to this biotrophic pathogen is thus an important challenge for poplar breeders. Severe damages occur through decreased photosynthesis efficiency, early defoliation and increased susceptibility to other pests and diseases. The PGF is sequencing this organism by the whole genome shotgun (WGS) method. Generally we make genomic libraries containing 3 different insert sizes. For all libraries, the DNA is randomly sheared, fragments end repaired and size selected prior to cloning. After cleanup the 3kb insert is ligated into pUC18 vector and the 8kb and 40kb fragments are ligated into pMCL200 and pcc1Fos vectors. The libraries are sequenced to a depth of approximately 4X, 4X, and 0.5-1X coverage respectively. The JGI has recently sequenced the genome to approximately 4x coverage of this plant pathogen, and is currently sequencing to the 8-9X depth prior to final assembly. The initial size was estimated to be approximately 61 Million bases (Mb), and analysis at the 4X assembly allowed us to revise this estimate to approximately 95 Mb. Library construction, genome assembly and size analysis will be presented.

The US Department of Energy's Joint Genome Institute is a high-throughput sequencing center and user facility that has sequenced a large number of microbial genomes. The strategy for most projects calls for construction of whole genome shotgun libraries from high-molecular weight DNA isolated from an axenic culture. In general, the JGI produces 3 insert size-selected libraries for all whole genome shotgun projects. We generate a 3kb high-copy pUC18 library, an 8kb low-copy pMCL200 library, and a 40kb pCC1FOS fosmid library. The DNA is randomly sheared, fragments are end-repaired for blunt-end cloning, and then size selected on an agarose gel, extracted and purified. 3 & 8kb inserts are cloned into the appropriate vector and transformed into E. coli. 40kb inserts are cloned, packaged and infected by phage into E. coli. PCR using primers flanking the inserts are used to determine the percentage of clones with inserts for both the 3 and 8kb libraries, before proceeding to production sequencing. Clones (10-384-well plates) from each of the 3 & 8kb libraries are initially sequenced and library quality is assessed at this stage before full sequencing is completed. Both 3 & 8kb libraries are sequenced to 4x sequencing coverage and the 40kb library is sequenced to 30x clone coverage. The 3 library approach generally results in more complete genome coverage at the draft stage, and pairing information allows for contig order and orientation and repeat resolution in the sequence. Finishing using standard methods is also facilitated by this approach.

The Department of Energy's (DOE) Joint Genome Institute (JGI) is one of the major publicly funded high throughput sequencing centers. The current capacity of the Production Genomics Facility (PGF) in Walnut Creek, California is approximately three billion bases per month and this year will generate up to 50 million lanes. JGI sequencing projects are initiated through several programs (http://www.jgi.doe.gov/programs/index.html). The two main programs for peer review of genome project proposals are the Community Sequencing Program (CSP) and the DOE Microbial Program. This year, the JGI processed a collection of DOE mission relevant sequencing projects ranging from prokaryotes to eukaryotes as well as several microbial communities. Data is released publicly on the JGI website and deposited in Genbank for all projects. An array of quality control measures and metrics has been put in place to evaluate projects prior to large scale sequencing to ensure efficient and timely completion of projects. This poster will present a menu of current JGI sequencing projects and describe the process of how projects are scheduled through the sequencing line, tracked and assessed for quality.