title: A robust PCR primer design platform applied to the detection of Acidobacteria Group 1 in soil.
abstract: 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 CO(2). 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.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA. jgans@lanl.gov
authors.forename: Jason D
authors.initials: JD
authors.lastname: Gans
authors.forename: John
authors.initials: J
authors.lastname: Dunbar
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Eichorst
authors.forename: La Verne
authors.initials: LV
authors.lastname: Gallegos-Graves
authors.forename: Murray
authors.initials: M
authors.lastname: Wolinsky
authors.forename: Cheryl R
authors.initials: CR
authors.lastname: Kuske
mesh_terms: D061271:Acidobacteria / Q000235:genetics / Q000302:isolation & purification*; D000465:Algorithms; D017931:DNA Primers / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012984:Software; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S