A robust PCR primer design platform applied to the detection of Acidobacteria Group 1 in soil

Gans, Jason D.; Dunbar, John; Eichorst, Stephanie A.; Gallegos-Graves, La Verne; Wolinsky, Murray; Kuske, Cheryl R.

doi:10.1093/nar/gks238

A robust PCR primer design platform applied to the detection of Acidobacteria Group 1 in soil

Journal Article · Tue Mar 20 04:00:00 EDT 2012 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gks238· OSTI ID:1625503

Gans, Jason D. ^[1]; Dunbar, John ^[2]; Eichorst, Stephanie A. ^[2]; Gallegos-Graves, La Verne ^[2]; Wolinsky, Murray ^[2]; Kuske, Cheryl R. ^[2]

Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Bioscience Division; DOE/OSTI
Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Bioscience Division

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.

View Accepted Manuscript (DOE)

Research Organization:: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC52-06NA25396

OSTI ID:: 1625503

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 12 Vol. 40; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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