Sequence analysis of 16S rRNA provides the acceptable basis for considering phylogenetic relationships. Nevertheless inhibitor Cisplatin the FAME analysis provides a convenient method with which to confirm the identity of the organism as it is maintained and studied over time. Growth conditions and DNA isolation For the preparation of genomic DNA, one of several colonies surrounded by a clear zone was picked from an agar plate (0.1% oat spelt xylan/ 0.1% yeast extract/ Zucker-Hankin medium [2], and grown in Zucker-Hankin/1% yeast extract at 30��C with shaking at 240 rpm. A culture (8 ml) at 0.6 OD600nm was inoculated into 48 ml of culture media (Zucker-Hankin, 1% yeast extract). The latter was grown to 0.6 OD600nm and cells were collected by centrifugation. High molecular weight DNA was prepared from these cells as per the protocol provided by JGI.

Cells were suspended in TE buffer (10 mM Tris-HCl, 1.0 mM EDTA), pH 8.0 and treated with lysozyme to lyse the cell wall. SDS and Proteinase K were added to denature and degrade proteins. NaCl and CTAB were added to facilitate subsequent precipitation. Cell lysates were extracted with phenol and chloroform and the DNA was precipitated by addition of isopropanol. The nucleic acid pellet was washed with 70% ethanol, dissolved in water and then treated with RNase A. Genome sequencing and assembly The genome of Pjdr2 was sequenced at the JGI using a combination of 8 kb and 40 kb (fosmid) DNA libraries. In addition to Sanger sequencing, 454 pyrosequencing [28] was performed to a depth of 20�� coverage.

All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [29]. Draft assemblies were based on 39,689 total reads. All three libraries provided 5.1�� coverage of the genome. The Phred/Phrap/Consed software package [30] was used for sequence GSK-3 assembly and quality assessment [31-33]. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher [34] or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walk, or PCR amplification (Roche Applied Science, Indianapolis, IN). A total of 1,028 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed sequence analysis of Pjdr2 contained 45,057 reads, achieving an average of 5.5-fold sequence coverage per base, with an error rate less than 1 in 100,000. The complete nucleotide sequence of Paenibacillus sp. strain JDR-2 and its annotation can be found online at the IMG (Integrated Microbial Genome) portal of JGI [35], as well as at the genome resource site of NCBI [36].