The Production Quality Control (QC) produces a series of assemblies, screening shotgun reads for contamination by vector sequence, for low-quality reads, and, in the case of microbial projects, for contamination by eukaryotic sequence. In coordination with the Microbial Finishing group lead at the PGF, an assembly that is "good enough" is named the Quality-controlled Draft (QD) assembly. Once the QD assembly is produced, the project passes from Production QC to one of the finishing groups at the JGI. During the finishing process, misassemblies are resolved, gaps between contigs are closed, and confirming reads are gathered for thinly covered areas. Judging whether a project is ready for finishing is a complex task, and requires answering numerous questions, such as: Is sufficient read and clone coverage present? Will existing contig linking information (scaffolding information) allow the closing of gaps between contigs? Has high or low GC content contributed to difficult-to-sequence or difficult-to-clone areas? Do the libraries have unusual features (e.g., being absent from certain areas of the genome, or having widely-spread insert size distributions)? Are contig GC content distributions consistent with project GC content distribution? How many repeats are present in the genome? Have repeats contributed to misassemblies? In order to facilitate the answering of these questions, we undertook the QD Extension project. QD Extension enhances the existing microbial finishing pipeline with results from the Phrap, PGA and Arachne assemblers, with scaffold information, and with coherent, informative web reports. By providing such additional information in a browsable, web-based format, we help the Microbial Finishing group lead at PGF more accurately and quickly determine whether a project is ready for finishing. This project facilitated the identification of a set of metrics that help to shed light on the finishing task that lies ahead of the biologist assigned to finish a

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 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).

Shotgun sequencing and finishing of an isolate of the Spirochaeta aurantia M1 genome, a free-living nonpathogenic Spirochete, is in process at the Joint Genome Institute. S. aurantia M1 is being sequenced due to its proximity on the phylogenetic tree to bacteria present in the termite hindgut that were partially sequenced during a metagenomic project at the JGI ,and, for improving the understanding of pathogenic Spirocheates through comparative genomic studies. The assembly is currently in 3 contigs and most of the finishing has been done using fosmid templates to close gaps. The shotgun assembly is a combination of fosmid sequencing, a novel Sanger approach, 454 sequencing, and Solexa data. The novel Sanger approach takes advantage of a bulk cloneless library preparation method involving the amplification of long DNA fragments onto beads, by emulsion PCR, and the subsequent sorting of individual beads by flow cytometry for seeding the Sanger chemistry process.