e. high salt tolerance. While not being a part of the Genomic selleckchem Dasatinib Encyclopedia of Bacteria and Archaea (GEBA) project [26], sequencing of the type strain will nonetheless aid the GEBA effort. The genome project is deposited in the Genomes On Line Database [27] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the Center of Biotechnology (CeBiTec). A summary of the project information is shown in Table 2. Table 2 Genome sequencing project information Growth conditions and DNA isolation C. halotolerans strain YIM 70093T, DSM 44683, was grown aerobically in CASO broth (Carl Roth GmbH, Karlsruhe,Germany) at 30��C. DNA was isolated from ~ 108 cells using the protocol described by Tauch et al. 1995 [28].

Genome sequencing and assembly The genome was sequenced using a 454 sequencing platform. A standard 3k paired end sequencing library was prepared according to the manufacturers protocol (Roche). Pyrosequencing reads were assembled using the Newbler assembler v2.3 (Roche). The initial Newbler assembly consisted of 81 contigs in six scaffolds with an additional 26 lone contigs. Analysis of the six scaffolds revealed one to be an extrachromosomal element (plasmid pCha1), four to make up the chromosome with the remaining one to contain the four copies of the RRN operon which caused the scaffold breaks. The scaffolds were ordered based on alignments to the complete genomes of C. glutamicum [29] and C. efficiens [30] and subsequent verification by restriction digestion, Southern blotting and hybridization with a 16S rDNA specific probe.

The Phred/Phrap/Consed software package [31-34] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, gaps between contigs were closed by editing in Consed Dacomitinib (for repetitive elements) and by PCR with subsequent Sanger sequencing (IIT Biotech GmbH, Bielefeld, Germany). A total of 61 additional reactions were necessary to close gaps not caused by repetitive elements. To raise the quality of the assembled sequence, Illumina reads were used to correct potential base errors and increase consensus quality. A WGS library was prepared using the Illumina-Compatible Nextera DNA Sample Prep Kit (Epicentre, WI, U.S.A) according to the manufacturer’s protocol. The library was sequenced in an 80 bp single read GAIIx run, yielding 1,497,321 total reads. Together, the combination of the Illumina and 454 sequencing platforms provided 46.0�� coverage of the genome. Genome annotation Gene prediction and annotation were done using the PGAAP pipeline [35]. Genes were identified using GeneMark [36], GLIMMER [37], and Prodigal [38].