The complete genome sequence was finished in February 2010. The GenBank accession numbers are “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_013889″,”term_id”:”289207187″,”term_text”:”NC_013889″NC_013889 and “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_013930″,”term_id”:”290242889″,”term_text”:”NC_013930″NC_013930 http://www.selleckchem.com/products/PD-0332991.html for the chromosome and plasmid, respectively. The genome project is listed in the Genome OnLine Database (GOLD) [26] as project Gc01217. Sequencing was carried out at the Joint Genome Institute (JGI) Finishing was done by JGI-Los Alamos National Laboratory (LANL) and initial automatic annotation by JGI-Oak Ridge National Laboratory (ORNL). Table 2 Genome sequencing project information Growth conditions and DNA isolation Thioalkalivibrio sp.

K90mix was grown with 40 mM thiosulfate as an energy source in standard sodium carbonate-bicarbonate medium at pH 10 and 2 M Na+ [2] at 35oC with shaking at 200 rpm. The cells were harvested by centrifugation and stored at minus 80��C for DNA extraction. Genomic DNA was obtained using phenol-chloroform-isoamylalcohol (PCI) extraction. The genomic DNA was extracted using PCI and precipitated with ethanol. The pellet was dried under vacuum and subsequently dissolved in water. The quality and quantity of the extracted DNA was evaluated using the DNA Mass Standard Kit provided by the JGI. Genome sequencing and assembly The genome of Thioalkalivibrio sp. K90mix was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [27].

Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 3,292 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the PGA assembler. Possible mis-assemblies were corrected and gaps between contigs were closed by editing in Consed, by custom primer walks from sub-clones or PCR products. A total of 181 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence.

Illumina reads were used to improve the final consensus quality using an in-house developed tool (the ‘Polisher’ [28]). The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Sanger and 454 sequencing platforms provided 42.1�� coverage of the Cilengitide genome. The final assembly contains 28,443 Sanger reads (10.0��) and 419,015 pyrosequencing reads (32.1��). Genome annotation Genes were identified using Prodigal [29] as part of the Oak Ridge National Laboratory genome annotation pipeline followed by a round of manual curation using the JGI GenePRIMP pipeline [30].