Sequencing, finishing and annotation were performed by the JGI A

Sequencing, finishing and annotation were performed by the JGI. A summary of the project PR-171 information is shown in Table 2. Table 2 Genome sequencing project information for Bradyrhizobium sp. strain WSM471. Growth conditions and DNA isolation Bradyrhizobium sp. strain WSM471 was grown to mid logarithmic phase in TY rich medium [24] on a gyratory shaker at 28��C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [25]. Genome sequencing and assembly The genome of Bradyrhizobium sp. WSM471 was generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina [26] and 454 technologies [27]. An Illumina GAii shotgun library which generated 67,039,982 reads totaling 5,095 Mb and 1 paired end 454 library with an average insert size of 5 Kb which generated 397,976 reads totaling 83.

7 Mb of 454 were generated for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [25]. The initial draft assembly contained 236 contigs in 2 scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data was assembled with Velvet, version 1.0.13 [28], and the consensus sequence were computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina Velvet consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS – 4.

24 (High Performance Software, LLC). The software Consed [29-31] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [32], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 327 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The estimated genome size is 7.

8 Mb and the final assembly is Brefeldin_A based on 53.8 Mb of 454 draft data which provides an average 6.9�� coverage of the genome and 4,879.9 Mb of Illumina draft data which provides an average 625.6�� coverage of the genome. Genome annotation Genes were identified using Prodigal [33] as part of the DOE-JGI Annotation pipeline [34] followed by a round of manual curation using the JGI GenePRIMP pipeline [35]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases.

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