Whole-Genome Assemblies of 56 Burkholderia Species

Burkholderia is a genus of betaproteobacteria that includes three notable human pathogens: B. cepacia, B. pseudomallei, and B. mallei. While B. pseudomallei and B. mallei are considered potential biowarfare agents, B. cepacia infections are largely limited to cystic fibrosis patients. Here, we present 56 Burkholderia genomes from 8 distinct species.

that was previously considered part of the genus Pseudomonas (1). Of the 82 recognized Burkholderia species, three are notable human pathogens: B. pseudomallei, B. mallei, and B. cepacia (an opportunistic lung infection pathogen in cystic fibrosis patients).
B. pseudomallei, the causative agent of melioidosis, is an environmental bacterium commonly found in southeastern Asia and northern Australia, where it causes multiple annual outbreaks. B. mallei is an obligate pathogen that primarily causes disease in horses, mules, and donkeys (called farcy) but is also the causative agent of glanders in humans, which can be either an acute or chronic infection (1). Both B. pseudomallei and B. mallei are on the CDC category B list due to their low infectious dose and high mortality rates, even with antibiotic therapy (2). While no evidence exists for the weaponization of B. pseudomallei, B. mallei has been developed as a weapon (2), and with their high transmission and mortality rates, both are considered potential biowarfare agents (3)(4)(5).
We sequenced the genomes of two Burkholderia cenocepacia, four B. cepacia, one Burkholderia gladioli, eight B. mallei, two Burkholderia multivorans, one Burkholderia oklahomensis, 34 B. pseudomallei, two Burkholderia thailandendsis, one Burkholderia xenovorans, and one strain not identified to species level. These additions will increase the publicly available scaffolded and completed genomes by 11 to 100% for each species (46% over all species).
High-quality genomic DNA was extracted from 100-ml bacterial cultures of purified isolates for each strain using the Qiagen Genomic tip-500, per the manufacturer's recommendations, with one minor variation. For biosafety level 3 (BSL3) Burkholderia organisms, all cultures were lysed overnight to ensure the sterility of the resulting extracted material. If sterility was not achieved, the nucleic acid was passed through a 0.45-M filter and rechecked for viable organisms before removal from the BSL3 suite.
The sequence data for each draft genome include at least two data types: Illumina (6), 454 (7), and for some, PacBio (8) tech-nologies. The draft genome coverages for each data type are included in the NCBI submission records; however, the Illumina (either unpaired or short-insert) coverages ranged from 114-to 1,067-fold, and the 454-based long-insert (insert size range, 5.2 to 12.7 kb) coverages were generally Ͻ50-fold. The combined draft data had genome coverages between 134-and 1,186-fold. The 454 and Illumina data were assembled together using Newbler and Velvet. All draft data were assembled together with AllPaths (9), and if the PacBio data were available and at Ն100ϫ coverage, they were assembled using HGAP (10). The consensus sequences from all assemblers were computationally shredded and assembled with a subset of read pairs from the long-insert library using Phrap (11,12). The resulting assemblies were manually and computationally improved using Consed (13) and in-house scripts.
The annotations were completed using the Ergatis workflow manager (14) and in-house scripts. The genomes are available in NCBI, and the raw data can be provided upon request. In-depth comparative analyses of these and other genomes will be published in subsequent reports.
Nucleotide sequence accession numbers. The genome accession numbers to public databases are listed in Table 1.

ACKNOWLEDGMENTS
Funding for this effort was provided by the Defense Threat Reduction Agency's Joint Science and Technology Office (DTRA J9-CB/JSTO). This paper is approved by the LANL for unlimited release (LA-UR-14-25908).
The views expressed in this article are those of the authors and do not necessarily reflect the official policy or positions of the Department of the Navy, Department of Defense, or the U.S. Government.