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 from a diversity of organisms and environmental communities. Often times, the amount of genomic DNA provided for library construction is very limited. It is imperative to develop a protocol to minimize the amount of genomic DNA required for library construction for the second-generation of sequencing platforms. We have begun constructing Roche/454 Shotgun and Illumina Paired End libraries with less than 1ug of genomic DNA by altering two key components from the standard operating protocol for library construction. The two key components that help minimize loss of genomic DNA are: shearing DNA via Covaris Adaptive Focused Acoustics(TM) (AFA) process instead of nebulization and utilizing Agencourt(R) AMPure(R) purification system to purify and select the size range of DNA fragments from contaminants and enzymes, with minimal loss of sample. This approach enables us to create Roche/454 libraries with as little as 300ng of genomic DNA and Illumina libraries with only 1ng of genomic DNA as the starting material.

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 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.