Determination of microbial diversity in environmental samples: pitfalls of PCR‐based rRNA analysis
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TL;DR: Specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis are covered.
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Abstract: After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
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Citations
Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora points to a strong assocation between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis
Rita Verhelst,Hans Verstraelen,Geert Claeys,Gerda Verschraegen,Joris R. Delanghe,Leen Van Simaey,Catharine De Ganck,Marleen Temmerman,Mario Vaneechoutte +8 more
- 01 Jan 2004
TL;DR: In this paper, the authors reported on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora, which indicated that much is to be learned about the composition of the vaginal micro-flora and its relation to the etiology of BV.
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Quantitative PCR provides a simple and accessible method for quantitative microbiota profiling.
Ching Jian,Panu K. Luukkonen,Panu K. Luukkonen,Hannele Yki-Järvinen,Hannele Yki-Järvinen,Anne Salonen,Katri Korpela +6 more
TL;DR: How quantitative PCR (qPCR) done in parallel to NGS library preparation provides an accurate estimation of absolute taxon abundances from NGS data and hence provides an attainable solution to compositionality in high-throughput microbiome analyses is described.
Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing.
TL;DR: It is suggested that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.
289
Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example
TL;DR: It is observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.
284
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