maritima MH2T, DSM 10411, was grown selleck chemicals llc anaerobically in medium 554 (HIPPEA medium) [21] at 55��C. DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol as recommended by the manufacturer with the following modification to improve cell lysis: additional 20��l lysozyme (100mg/��l) and 10��l mutalysin were used for 30 min incubation at 37��C, followed by three hours incubation at 58��C with 20��l proteinase K. DNA is available through the DNA Bank Network [22]. 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 [23]. Pyrosequencing reads were assembled using the Newbler assembler (Roche).
The initial Newbler assembly, consisting of 70 contigs in one scaffold, was converted into a phrap [24] assembly by making fake reads from the consensus to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (4,403.8 Mb) was assembled with Velvet [25] 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 66.2 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 [24] 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 [23], Dupfinisher [26], or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 357 additional reactions and one shatter library 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 [27]. 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 1,241.
6 �� coverage of the genome. The final assembly contained 112,403 pyrosequence and 57,283,044 Illumina reads. Genome annotation Genes were identified using Prodigal [28] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the Carfilzomib JGI GenePRIMP pipeline [29]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases.