Title |
16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles
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Published in |
Microbiome, September 2014
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DOI | 10.1186/2049-2618-2-31 |
Pubmed ID | |
Authors |
Jun Hang, Valmik Desai, Nela Zavaljevski, Yu Yang, Xiaoxu Lin, Ravi Vijaya Satya, Luis J Martinez, Jason M Blaylock, Richard G Jarman, Stephen J Thomas, Robert A Kuschner |
Abstract |
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. |
X Demographics
Geographical breakdown
Country | Count | As % |
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United States | 1 | 10% |
Japan | 1 | 10% |
Mexico | 1 | 10% |
Australia | 1 | 10% |
Unknown | 6 | 60% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 4 | 40% |
Scientists | 3 | 30% |
Science communicators (journalists, bloggers, editors) | 2 | 20% |
Practitioners (doctors, other healthcare professionals) | 1 | 10% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
United States | 5 | 3% |
Japan | 2 | 1% |
Argentina | 2 | 1% |
Brazil | 2 | 1% |
United Kingdom | 1 | <1% |
Canada | 1 | <1% |
Germany | 1 | <1% |
Switzerland | 1 | <1% |
Chile | 1 | <1% |
Other | 2 | 1% |
Unknown | 176 | 91% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Researcher | 42 | 22% |
Student > Master | 35 | 18% |
Student > Ph. D. Student | 30 | 15% |
Student > Bachelor | 22 | 11% |
Student > Doctoral Student | 12 | 6% |
Other | 32 | 16% |
Unknown | 21 | 11% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 23 | 12% |
Medicine and Dentistry | 18 | 9% |
Immunology and Microbiology | 11 | 6% |
Engineering | 6 | 3% |
Other | 17 | 9% |
Unknown | 29 | 15% |