|
| Status |
Public on Jun 27, 2013 |
| Title |
Ba_1000_Aerosol_rep_2 |
| Sample type |
genomic |
| |
|
| Source name |
Aerosol filter sample spiked with B. anthracis DNA
|
| Organism |
Bacillus anthracis str. Ames |
| Characteristics |
ba copies: 1000
background: Aerosol
rep: 2
|
| Treatment protocol |
B. anthracis Ames DNA was acquired from the LLNL select agent laboratory and confirmed to be free of viable cells or spores by plating of 1/10 the total DNA volume on blood agar plates followed by incubation at 37°C for 72 hours. DNA concentration was determined by measurement using a Qubit fluorometer and the number of femtograms/genome equivalent was determined based on GenBank chromosomal and plasmid genome sizes. B. anthracis genomic DNA was added to nucleic acid extracted from the two environmental sources in six amounts (1, 10, 100, 1000, 10000, and 100000 genomic equivalents of B. anthracis DNA). B. anthracis DNA was mixed with 100 pg of DNA extracted from aerosol filters or 1 ng DNA extracted from the combined Oakland and San Francisco soil extracts.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from soil using the UltraClean Soil DNA Isolation Kit (MoBio, Carlsbad, CA) using the manufacturer’s alternative protocol for maximum yield. Following extraction, 1 ng of each extracted DNA was used in a real-time PCR assay to test for inhibition. All samples showed a high level of PCR inhibition and were reprocessed starting from Step 12 of the MoBio alternative protocol, intended to remove excess humic acid. For aerosol samples, primary sampling filters were obtained from BioWatch aerosol collection units (collected April 2009) from the National Capital Region Laboratory. Filters were added to 30 mL 100 mM phosphate buffer (pH 7.4) containing 0.05% (v/v) Tween 80 and vortexed for 30 seconds followed by incubation on a rocking shaker for 15 minutes. This agitation procedure was repeated three times. The filters were removed from the tube and the remaining solution centrifuged at 3200 x g for 30 minutes at 4oC. Following centrifugation, the supernatant was removed and discarded. DNA purification was subsequently performed using the UltraClean Soil DNA Isolation Kit (MoBio). DNA concentrations were measured using a Qubit fluorometer (Life Technologies, Grand Island, NY).
|
| Label |
Cy3
|
| Label protocol |
B. anthracis-spiked samples were amplified using the REPLI-g Midi Kit (Qiagen, Gaithersburg, MD), intended to provide uniform whole genome amplification. This kit was used according to the manufacturer’s instructions, allowing samples to amplify for 16 hours at 30°C. Amplified samples were purified using the Qiaquick PCR Purification Kit (Qiagen), and yielded 17.0 ± 1.8 µg amplified DNA for each sample. Amplified environmental DNA spiked with B. anthracis Ames genomic DNA was fluorescently labeled using the NimbleGen One-Color DNA Labeling Kit (Roche) according to the manufacturer’s recommended protocols.
|
| |
|
| Hybridization protocol |
DNA was hybridized to the census array using the NimbleGen hybridization kit. Samples were allowed to hybridize for 17 hours and washed using the NimbleGen Wash Buffer Kit (Roche).
|
| Scan protocol |
Microarrays were scanned on an Axon GenePix 4000B 5 µm scanner (Molecular Devices, Sunnyvale, CA).
|
| Description |
data used for Supplementary Table S6
|
| Data processing |
Data was analyzed using NimbleScan software and custom software developed at Lawrence Livermore National Laboratory, CA, USA and described in 'Gardner et al. A Micobial Detection Array (MDA) for Viral and Bacterial Detection. BMC Genomics 2010 Nov 25;11:668'. Additional stringency criteria were applied to exclude bacterial sequences and viruses having fewer than 20% of probes detected out of those expected. Probes were classified as detected if the intensity exceeded a threshold equal to the 99th percentile of intensities for negative control probes.
|
| |
|
| Submission date |
Jun 26, 2013 |
| Last update date |
Jun 27, 2013 |
| Contact name |
Kevin McLoughlin |
| E-mail(s) |
mcloughlin2@llnl.gov
|
| Phone |
(925)423-5486
|
| Organization name |
Lawrence Livermore National Laboratory
|
| Department |
Global Security
|
| Lab |
Pathogen Bioinformatics
|
| Street address |
PO Box 808
|
| City |
Livermore |
| State/province |
CA |
| ZIP/Postal code |
94551 |
| Country |
USA |
| |
|
| Platform ID |
GPL17345 |
| Series (1) |
| GSE48316 |
Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing [NimbleGen Array data] |
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