UDP-sugars are present in cells of strain 1AT [40]. The structures of the two major UDP-sugars are identified as UDP-��-GlcNAc3NAc and UDP-��-GlcNAc3NAc-(4��1)-��-GlcpNAc3NAc [40]. UDP-sugars are intermediates of an N-linked glycosylation pathway of strain 1AT [40]. Strain selleck inhibitor 1AT performs a posttranscriptional modification of transfer RNA [38]. Genome sequencing and annotation Genome project history This organism was selected for sequencing on the basis of its phylogenetic position [41], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [42]. The genome project is deposited in the Genome On Line Database [14] 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 P. fumarii 1AT, DSM 11204, was grown anaerobically in DSMZ medium 792 (Pyrolobus fumarii medium) [43] at 103��C. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the standard protocol as recommended by the manufacturer. 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 [44]. Pyrosequencing reads were assembled using the Newbler assembler (Roche).

The initial Newbler assembly consisting of ten contigs in one scaffold was converted into a phrap [45] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina sequencing data (3,232.0 Mb) was assembled with Velvet [46] 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 79.2 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [45] 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 [44], Dupfinisher, or sequencing cloned bridging PCR fragments Dacomitinib with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [47]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 12 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 [48].