Accession Number:

ADA610745

Title:

16S rRNA Gene Pyrosequencing of Reference and Clinical Samples and Investigation of the Temperature Stability of MicroBiome Profiles

Descriptive Note:

Journal article

Corporate Author:

WALTER REED ARMY INST OF RESEARCH SILVER SPRING MD

Report Date:

2014-09-16

Pagination or Media Count:

17.0

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 201HMP202 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 swab

Subject Categories:

  • Medicine and Medical Research
  • Microbiology

Distribution Statement:

APPROVED FOR PUBLIC RELEASE