Dataset Information

A direct PCR approach to accelerate analyses of human-associated microbial communities.

ABSTRACT: Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.

SUBMITTER: Flores GE

PROVIDER: S-EPMC3433448 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A direct PCR approach to accelerate analyses of human-associated microbial communities.

Flores Gilberto E GE Henley Jessica B JB Fierer Noah N

PloS one 20120904 9

Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach ...[more]

PMID: 22962617

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Project description:Daqu made from raw wheat, barley or pea is used as an inoculum for the fermentation of Chinese Baijiu. In this study, the microbial communities of four different types of Daqus (sauce-flavor Wuling Daqu, sauce and strong-flavor Baisha Daqu, strong-flavor Deshan Daqu, and light-flavor Niulanshan Daqu) were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), phospholipid fatty acid (PLFA) analysis, and Biolog EcoPlates analysis (Biolog). Clear differences were seen between the microbial communities of the four Daqus. PCR-DGGE showed differences in the number and brightness of bands between the Daqus, indicating the presence of unique bacterial species in Deshan Daqu, Wuling Daqu, and Niulanshan Daqu. Lactobacillus sanfranciscensis, Bacillus thermoamylovorans, and some unclassified bacteria were unique to Wuling Daqu, Deshan Daqu, and Niulanshan Daqu, respectively. Moreover, some bacterial species were observed in all four Daqus. A total of 26 PLFAs between C12 to C20 were detected from the four Daqus by PLFA analysis. Wuling Daqu had the highest total and fungal biomasses, Baisha Daqu had the highest bacterial biomass, and Niulanshan Daqu had the highest ratio of fungal biomass to bacterial biomass. The Biolog results indicated differences in the carbon source use and mode of the four Daqus, and also demonstrated that each Daqu had varying abilities to utilize different types of carbon sources. The cluster analysis of the three methods showed that the microbial communities of the four Daqus were different. This study also demonstrates the applicability of the three analytical methods in the evaluating of the microbial communities of Daqus.Daqu made from raw wheat, barley or pea is used as an inoculum for the fermentation of Chinese Baijiu. In this study, the microbial communities of four different types of Daqus (sauce-flavor Wuling Daqu, sauce and strong-flavor Baisha Daqu, strong-flavor Deshan Daqu, and light-flavor Niulanshan Daqu) were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), phospholipid fatty acid (PLFA) analysis, and Biolog EcoPlates analysis (Biolog). Clear differences were seen between the microbial communities of the four Daqus. PCR-DGGE showed differences in the number and brightness of bands between the Daqus, indicating the presence of unique bacterial species in Deshan Daqu, Wuling Daqu, and Niulanshan Daqu. Lactobacillus sanfranciscensis, Bacillus thermoamylovorans, and some unclassified bacteria were unique to Wuling Daqu, Deshan Daqu, and Niulanshan Daqu, respectively. Moreover, some bacterial species were observed in all four Daqus. A total of 26 PLFAs between C12 to C20 were detected from the four Daqus by PLFA analysis. Wuling Daqu had the highest total and fungal biomasses, Baisha Daqu had the highest bacterial biomass, and Niulanshan Daqu had the highest ratio of fungal biomass to bacterial biomass. The Biolog results indicated differences in the carbon source use and mode of the four Daqus, and also demonstrated that each Daqu had varying abilities to utilize different types of carbon sources. The cluster analysis of the three methods showed that the microbial communities of the four Daqus were different. This study also demonstrates the applicability of the three analytical methods in the evaluating of the microbial communities of Daqus.

Project description:Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.

Dataset Information

A direct PCR approach to accelerate analyses of human-associated microbial communities.

Publications

A direct PCR approach to accelerate analyses of human-associated microbial communities.

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