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Comparison of DNA extraction methods in analysis of salivary bacterial communities.

ABSTRACT: Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

SUBMITTER: Lazarevic V

PROVIDER: S-EPMC3701005 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Comparison of DNA extraction methods in analysis of salivary bacterial communities.

Lazarevic Vladimir V Gaïa Nadia N Girard Myriam M François Patrice P Schrenzel Jacques J

PloS one 20130703 7

Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a b ...[more]

PMID: 23844068

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Project description:ObjectivesGood-quality and sufficient DNA is essential for diagnostics and vaccine development. We aimed to compare six DNA extraction techniques applied to Loa loa microfilariae in order to evaluate the purity and integrity of extracts in terms of quality and quantity.MethodsThe microfilariae were purified via a Percoll gradient procedure with blood from hyper-microfilaremic individuals (> 30,000 microfilaria [mf]/ml). DNA extraction was carried out in duplicate at a rate of 350,000 mf/tube for each technique: phenol/chloroform, commercial Qiagen kit, salting out, Tris-EDTA, methanol, and cetyltrimethylammonium bromide (CTAB). The integrity, purity, concentration, and quality of the DNA extracts were successively verified by agarose gel electrophoresis, spectrophotometry (A260/A280 and A260/A230 wavelength ratio), Qubit fluorometry, and endonuclease and polymerase activity. The six techniques were compared on the basis of the following parameters: concentration, purity, efficiency, effectiveness, integrity, safety of the technique, as well as cost and duration of the protocol.ResultsThe ratios of the optical densities of the extracts A260/A280 and A260/A230 were, respectively: phenol/chloroform (1.82; 1.11), Qiagen (1.93; 1.36), salting-out (1.9; 2.04), Tris-EDTA (1.99; 1.183), methanol (2.126; 1.343), and CTAB (2.01; 2.426). The DNA yield was: phenol/chloroform (3.920 μg), Qiagen (10.280 μg), salting-out (10.390 μg), Tris-EDTA (0.5528 μg), methanol (0.1036 μg), and CTAB (1.115 μg). Endonuclease and polymerase activity was demonstrated by digestion of DNA and through amplicons obtained via polymerase chain reaction assays with phenol/chloroform, Qiagen, and salting-out extracts.ConclusionThe phenol/chloroform, Qiagen, and salting-out DNA extracts were all of good quality. Salting out had the best yield followed by Qiagen and then phenol/chloroform. Endonuclease and polymerase activity was effective in all three extracts despite the presence of some contaminants. These methods are therefore suitable for the extraction of DNA from Loa loa microfilariae. Tris-EDTA and methanol did not show adequate sensitivity, while CTAB was found to be unsuitable.

Project description:BackgroundDNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S).ResultsOverall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did.ConclusionsMuseum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The difference between extraction methods was minimal and we recommend using an established kit for a first attempt because experience and routine in handling might be more important than slight performance differences of the various kits. Finally, we identify fixation, storage, sample conservation and documentation of the specimens' history rather than the DNA extraction method to be the most crucial factors for PCR success.

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Comparison of DNA extraction methods in analysis of salivary bacterial communities.

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Comparison of DNA extraction methods in analysis of salivary bacterial communities.

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