Genome sequencing and annotation

Genome project history
This organism was selected for sequencing on the basis of the DOE Joint Genome Institute Community Sequencing Program (CSP) 2008. The genome project is deposited in the Genomes On Line Database [39] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Table 2  Genome sequencing project information

Growth conditions and DNA isolation
Strain ATCC 8093T was grown from a culture of DSMZ 506 in DSMZ medium 69 at 28°Cg DNA was purified using the Genomic-tip 100 System (Qiagen) following the directions provided by the supplier. The purity, quality and size of the bulk gDNA preparation were assessed by JGI according to DOE-JGI guidelines.

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [57]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 13 contigs in one scaffold was converted into a phrap [58] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (211.3 Mb) were assembled with Velvet [59] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 259.9 Mb 454 draft data and all of the 454 paired-end data. Newbler parameters were -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [58] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [58], Dupfinisher [60], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 43 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [61]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 97.8 × coverage of the genome. The final assembly contained 865,253 pyrosequence and 6,036,863 Illumina reads.

Genome annotation
Genes were identified using Prodigal [62] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [63]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [64, RNAMMer [65], Rfam [66], TMHMM [67], and SignalP [68].