Complete Genome Sequences for 35 Biothreat Assay-Relevant Bacillus Species
This article has been corrected.
VIEW CORRECTIONABSTRACT
In 2011, the Association of Analytical Communities (AOAC) International released a list of Bacillus strains relevant to biothreat molecular detection assays. We present the complete and annotated genome assemblies for the 15 strains listed on the inclusivity panel, as well as the 20 strains listed on the exclusivity panel.
GENOME ANNOUNCEMENT
For several years the idea that biothreat and contamination detection methods need to be better characterized has been discussed (1–3). This led the Association of Analytical Communities (AOAC) International to compose a bacterial strain list for evaluation when designing molecular detection assays. This list, termed the Stakeholder Panel on Agent Detection Assays (SPADA), includes 15 inclusivity and 20 exclusivity Bacillus strains (4). As testing pertaining to these strains involves nucleic acid analyses, complete genome assemblies can further improve assays confidence, a major issue in both positive and negative results (5). Here, we describe complete genomes for all 35 strains.
Each microbial isolate genome was assembled using at least two data sets (specific data types and coverages are listed in the NCBI records): Illumina (short- and/or long-insert paired data), Roche 454 (long-insert paired data), and PacBio long reads. Short- and long-insert paired data were assembled in both Newbler and Velvet and computationally shredded into 1.5-kbp overlapping shreds. If PacBio coverage was 100× or greater, the data were assembled using PacBio's Hierarchical Genome Assembly Process (HGAP) (6), all data were additionally assembled together in Allpaths (7). Consensus sequences from HGAP and Allpaths were computationally shredded into 10-kbp overlapping pieces. All shreds were integrated using Phrap. Possible misassemblies were corrected and repeat regions verified using in-house scripts and Consed for manual editing (8–10). All but one of the genomes were assembled into finished-quality complete genomes (11). Each genome assembly was annotated using an Ergatis-based (12) workflow with minor manual curation.
Genome assemblies range from 3.4 to 6.7 Mb (Table 1; smallest B. coagulans DSM 1 and largest B. thuringiensis subsp. Morrisoni HD 600) with up to 14 plasmids (mean, 2.9 ± 0.5) and G+C contents of 33 to 47% (only B. coagulans DSM 1 has a G+C content greater than 40%).
TABLE 1
| Strain | Accession no. | Panel | AOAC no. | Assembly (bp) | No. of plasmids | G+C content (%) |
|---|---|---|---|---|---|---|
| B. anthracis | ||||||
| Turkey32 | CP009314–CP009316 | I | BA15 | 5,505,298 | 2 | 35 |
| 2002013094 | CP009900–CP009902 | I | BA12 | 5,601,083 | 2 | 35 |
| Ames_BA1004 | CP009979–CP009981 | I | BA5 | 5,503,969 | 2 | 35 |
| BA1015 | CP009542–CP009544 | I | BA4 | 5,491,163 | 2 | 35 |
| BA1035 | CP009698–CP009700 | I | BA10 | 5,487,253 | 2 | 35 |
| Canadian bison | CP010320–CP010322 | I | BA1 | 5,505,775 | 2 | 35 |
| K3 | CP009329–CP009331 | I | BA6 | 5,504,993 | 2 | 35 |
| Ohio ACB | CP009339–CP009341 | I | BA7 | 5,498,337 | 2 | 35 |
| PAK-1 | CP009324–CP009325 | I | BA3 | 5,403,381 | 1 | 35 |
| Pasteur | CP009475–CP009476 | I | BA13 | 5,294,803 | 1 | 35 |
| RA3 | CP009695–CP009697 | I | BA11 | 5,489,869 | 2 | 35 |
| SK-102 | CP009462–CP009464 | I | BA8 | 5,505,681 | 2 | 35 |
| Sterne | CP009540–CP009541 | I | BA14 | 5,409,120 | 1 | 35 |
| V770-NP-1R | CP009597–CP009598 | I | BA2 | 5,410,397 | 1 | 35 |
| Vollum 1B | CP009326–CP009328 | I | BA9 | 5,506,626 | 2 | 35 |
| B. cereus | ||||||
| 03BB102 | CP009317–CP009318 | E | BANN13 | 5,448,107 | 1 | 35 |
| D17 | CP009299–CP009300 | E | BANN7 | 5,590,358 | 1 | 35 |
| 03BB108 | CP009634–CP009641 | E | BANN14 | 6,450,959 | 7 | 33 |
| 3A | CP009593–CP009596 | E | BANN2 | 5,642,300 | 3 | 35 |
| ATCC 4342 | CP009627–CP009628 | E | BANN10 | 5,306,298 | 1 | 35 |
| E33L | CP009965–CP009970 | E | BANN6 | 5,846,781 | 5 | 35 |
| FM1 | CP009368–CP009369 | E | BANN11 | 5,697,763 | 1 | 35 |
| G9241 | CP009589–CP009592 | E | BANN12 | 5,720,073 | 3 | 35 |
| S2-8 | CP009604–CP009606 | E | BANN1 | 5,642,468 | 3 | 35 |
| B. coagulans | ||||||
| ATCC 7050 | CP009709 | E | BANN18 | 3,366,995 | 0 | 47 |
| B. megaterium | ||||||
| ATCC 14581 | CP009915–CP009921 | E | BANN20 | 5,746,640 | 6 | 38 |
| B. mycoides | ||||||
| ATCC 6462 | CP009689–CP009692 | E | BANN19 | 5,637,053 | 3 | 35 |
| B. thuringiensis | ||||||
| Al. Hakam | CP009645–CP009651 | E | BANN9 | 5,676,963 | 6 | 36 |
| 97-27 | CP010087–CP010088 | E | BANN4 | 5,312,686 | 1 | 35 |
| HD 1011 | CP009332–CP009336 | E | BANN3 | 6,093,375 | 4 | 35 |
| HD 571 | CP009599–CP009600 | E | BANN8 | 5,312,179 | 1 | 35 |
| HD 682 | CP009717–CP009720 | E | BANN8 | 5,291,389 | 3 | 35 |
| subsp. Kurstaki HD 1 | CP009998–CP010012 | E | BANN16 | 6,859,374 | 14 | 35 |
| subsp. Morrisoni HD 600b | JTHH00000000 | E | BANN17 | 6,916,808 | 7 | 35 |
| subsp. thuringiensis HD 1002 | CP009344–CP009351 | E | BANN15 | 6,572,702 | 7 | 35 |
a
If a strain is listed in the inclusivity panel, it is notated with an “I”; if it is in the exclusivity panel, it is notated with an “E.”
b
Strain B. thuringiensis subsp. Morrisoni HD600 is at Improved High Quality Draft (IHQD) status in 8 contigs, while all other genomes are completed to finished status (11).
Nucleotide sequence accession numbers.
Accession numbers for all 35 genomes 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) and Department of Homeland Security Science and Technology Directorate, award HSHQDC-08-X-00790.
This manuscript is approved by LANL for unlimited release (LA-UR-14-29607).
The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, the Department of Defense, or the United States Government.
Bacterial strains were obtained from the Department of Defense's Unified Culture Collection (http://www.usamriid.army.mil/ucc/).
REFERENCES
1.
Sanderson WT, Hein MJ, Taylor L, Curwin BD, Kinnes GM, Seitz TA, Popovic T, Holmes HT, Kellum ME, McAllister SK, Whaley DN, Tupin EA, Walker T, Freed JA, Small DS, Klusaritz B, Bridges JH. 2002. Surface sampling methods for Bacillus anthracis spore contamination. Emerg Infect Dis 8:1145–1151.
2.
Rhodes K. 2005. Anthrax detection: agencies need to validate sampling activities in order to increase confidence in negative results. U.S. Government Accountability Office, Washington, DC.
3.
Calfee MW, Lee SD, Ryan SP. 2013. A rapid and repeatable method to deposit bioaerosols on material surfaces. J Microbiol Methods 92:375–380.
4.
Association of Analytical Communities International. 2011. AOAC SMPR 2010.003: standard method performance requirements for polymerase chain reaction (PCR) methods for detection of Bacillus anthracis in aerosol collection filters and/or liquids. J AOAC Int 94:1347–1351.
5.
Morrow JB, Davenport M. 2012. Standards activities to support a biothreat mission capability in the United States. EuroReference 7:43–44.
6.
Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569.
7.
Butler J, MacCallum I, Kleber M, Shlyakhter IA, Belmonte MK, Lander ES, Nusbaum C, Jaffe DB. 2008. ALLPATHS: de novo assembly of whole-genome shotgun microreads. Genome Res 18:810–820.
8.
Ewing B, Green P. 1998. Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res 8:186–194.
9.
Ewing B, Hillier L, Wendl MC, Green P. 1998. Base-calling of automated sequencer traces UsingPhred. I. Accuracy assessment. Genome Res 8:175–185.
10.
Gordon D, Abajian C, Green P. 1998. Consed: a graphical tool for sequence finishing. Genome Res 8:195–202.
11.
Chain PS, Grafham DV, Fulton RS, FitzGerald MG, Hostetler J, Muzny D, Ali J, Birren B, Bruce DC, Buhay C, Cole JR, Ding Y, Dugan S, Field D, Garrity GM, Gibbs R, Graves T, Han CS, Harrison SH, Highlander S, Hugenholtz P, Khouri HM, Kodira CD, Kolker E, Kyrpides NC, Lang D, Lapidus A, Malfatti SA, Markowitz V, Metha T, Nelson KE, Parkhill J, Pitluck S, Qin X, Read TD, Schmutz J, Sozhamannan S, Sterk P, Strausberg RL, Sutton G, Thomson NR, Tiedje JM, Weinstock G, Wollam A, Genomic Standards Consortium Human Microbiome Project Jumpstart Consortium, Detter JC. 2009. Genome project standards in a new era of sequencing. Science 326:236–237.
12.
Hemmerich C, Buechlein A, Podicheti R, Revanna KV, Dong Q. 2010. An Ergatis-based prokaryotic genome annotation Web server. Bioinformatics 26:1122–1124.
Information & Contributors
Information
Published In
Genome Announcements
Volume 3 • Number 2 • 30 April 2015
eLocator: 10.1128/genomea.00151-15
Copyright
© 2015 Johnson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
History
Received: 5 February 2015
Accepted: 4 March 2015
Published online: 30 April 2015
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