Journal
NUCLEIC ACIDS RESEARCH
Volume 40, Issue 12, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gks238
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Funding
- US Department of Homeland Security [HSHQDC-08X-00763, HSHQDC-07-X-00908, HSHQDC-08-X-00793]
- US Department of Energy
- Office of Biological and Environmental Research [2009LANLF260]
- US Department of Energy Laboratory Directed Research and Development program [LDRD-ER 20110665ER]
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Environmental biosurveillance and microbial ecology studies use PCR-based assays to detect and quantify microbial taxa and gene sequences within a complex background of microorganisms. However, the fragmentary nature and growing quantity of DNA-sequence data make group-specific assay design challenging. We solved this problem by developing a software platform that enables PCR-assay design at an unprecedented scale. As a demonstration, we developed quantitative PCR assays for a globally widespread, ecologically important bacterial group in soil, Acidobacteria Group 1. A total of 33 684 Acidobacteria 16S rRNA gene sequences were used for assay design. Following 1 week of computation on a 376-core cluster, 83 assays were obtained. We validated the specificity of the top three assays, collectively predicted to detect 42% of the Acidobacteria Group 1 sequences, by PCR amplification and sequencing of DNA from soil. Based on previous analyses of 16S rRNA gene sequencing, Acidobacteria Group 1 species were expected to decrease in response to elevated atmospheric CO2. Quantitative PCR results, using the Acidobacteria Group 1-specific PCR assays, confirmed the expected decrease and provided higher statistical confidence than the 16S rRNA gene-sequencing data. These results demonstrate a powerful capacity to address previously intractable assay design challenges.
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