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Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

ABSTRACT: Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations.

SUBMITTER: Wagner AO

PROVIDER: S-EPMC4461152 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

Wagner Andreas O AO Praeg Nadine N Reitschuler Christoph C Illmer Paul P

Applied Soil Ecology. 20150901

Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields w ...[more]

PMID: 26339125

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Project description:BackgroundHigh-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities.ResultsPMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies.ConclusionsThis study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.

Project description:Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel virus of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of host genomic DNA (hgDNA) contamination. Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA but are cell membrane-impermeable and thus are unable to bind protected DNA and RNA such as viral genomic material. DNA modified by EMA or PMA is not amplifiable by polymerase. In order to assess the capacity of EMA or PMA to lower hgDNA, serum or lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with different combination of treatment: with or without ultracentrifugation and incubation with or without different concentration of EMA or PMA. PVDA and HTS were used to evaluate the capacity of both techniques to detect the presence of PRRSV from each sample. Negative results were obtained by PVDA and low amount of PRRSV specific reads were obtained by HTS with untreated samples or samples treated only by ultracentrifugation. An increase capacity of PRRSV detection was observable by PVDA in EMA and PMA treated samples but PVDA best results were obtained following PMA treatment, with or without ultracentrifugation. HTS sensitivity was also improved by a treatment with EMA or PMA, but the number of reads was significantly higher in PMA treated samples. These results support the use of PMA as a treatment to increase sensitivity of PVDA and HTS. A non specific DNA probe is used as a negative hybridization control. Also, specifics probes targeting pUC19 plasmid DNA is used as a DNA array positive control as well as a localization control. Finally, there are 34 probes of 70 nucleotides spotted in duplicate were selected to target PRRSV conserved regions. A total of 80 different tests were done by DNA array in order to evaluate different treatments conditions with EMA or PMA, with or without ultracentrifugation. A total of 58 samples were tested on specific lung tissue homogenates spiked with different concentration of PRRSV and 12 samples were serum samples spiked with one concentration of PRRSV. Finally. A total of six samples

Dataset Information

Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

Publications

Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

Similar Datasets

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