title: 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles.
abstract: BACKGROUND
  Sample storage conditions, extraction methods, PCR primers, and parameters are major factors that affect metagenomics analysis based on microbial 16S rRNA gene sequencing. Most published studies were limited to the comparison of only one or two types of these factors. Systematic multi-factor explorations are needed to evaluate the conditions that may impact validity of a microbiome analysis. This study was aimed to improve methodological options to facilitate the best technical approaches in the design of a microbiome study. Three readily available mock bacterial community materials and two commercial extraction techniques, Qiagen DNeasy and MO BIO PowerSoil DNA purification methods, were used to assess procedures for 16S ribosomal DNA amplification and pyrosequencing-based analysis. Primers were chosen for 16S rDNA quantitative PCR and amplification of region V3 to V1. Swabs spiked with mock bacterial community cells and clinical oropharyngeal swabs were incubated at respective temperatures of -80°C, -20°C, 4°C, and 37°C for 4 weeks, then extracted with the two methods, and subjected to pyrosequencing and taxonomic and statistical analyses to investigate microbiome profile stability.
  
  
  RESULTS
  The bacterial compositions for the mock community DNA samples determined in this study were consistent with the projected levels and agreed with the literature. The quantitation accuracy of abundances for several genera was improved with changes made to the standard Human Microbiome Project (HMP) procedure. The data for the samples purified with DNeasy and PowerSoil methods were statistically distinct; however, both results were reproducible and in good agreement with each other. The temperature effect on storage stability was investigated by using mock community cells and showed that the microbial community profiles were altered with the increase in incubation temperature. However, this phenomenon was not detected when clinical oropharyngeal swabs were used in the experiment.
  
  
  CONCLUSIONS
  Mock community materials originated from the HMP study are valuable controls in developing 16S metagenomics analysis procedures. Long-term exposure to a high temperature may introduce variation into analysis for oropharyngeal swabs, suggestive of storage at 4°C or lower. The observed variations due to sample storage temperature are in a similar range as the intrapersonal variability among different clinical oropharyngeal swab samples.
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Hang
authors.affiliation: Department of Defense Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, MD 21702, USA.
authors.forename: Valmik
authors.initials: V
authors.lastname: Desai
authors.affiliation: Department of Defense Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, MD 21702, USA.
authors.forename: Nela
authors.initials: N
authors.lastname: Zavaljevski
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Lin
authors.affiliation: Department of Defense Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, MD 21702, USA ; Current address: Qiagen Sciences Inc, Frederick, MD 21703, USA.
authors.forename: Ravi Vijaya
authors.initials: RV
authors.lastname: Satya
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Luis J
authors.initials: LJ
authors.lastname: Martinez
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA ; Current address: Infectious Diseases Service, WRNMMC, Bethesda, MD 20889, USA.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Blaylock
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Jarman
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Thomas
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kuschner