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Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples.

ABSTRACT: The anaerobic digestion (AD) of organic waste for biogas production has received much attention in recent years due to the increasing need for renewable energy and environmentally friendly waste management systems. Identification of the microbial community involved in AD aids in better understanding and optimising of the process. The choice of DNA extraction method is an integral step in any molecular biodiversity study. In the present study, potential biases introduced by DNA extraction methods were examined by comparing quality, quantity and representability of DNA extracted from AD samples using various extraction methods. In spite of the non-kit based method (cetyltrimethylammonium bromide) yielding the largest quantity of DNA (approximately 44 µg DNA per gram dry weight), the extracted DNA contained PCR inhibitors. Furthermore, the quantity of extracted DNA was not proportional to species diversity. Diversity, determined using denaturing gradient gel electrophoresis (DGGE), was strongly linked to the type of extraction method used. The spin-column filter-based kit that incorporated mechanical and chemical lysis (Macherey-Nagel kit) gave the best results in terms of bacterial and archaeal diversity (Shannon-Wiener indices: average 2.5 and 2.6, respectively). Furthermore, this kit was the most effective at lysing hard-to-lyse bacterial and archaeal cells. The choice of DNA extraction method significantly influences the reliability and comparability of results obtained during AD microbial ecology investigations. Moreover, the careful selection of the DNA extraction method is of particular importance when analysing AD samples since these samples are rich in PCR inhibitors and hard-to-lyse cells such as archaea and gram-positive bacteria.

SUBMITTER: Roopnarain A

PROVIDER: S-EPMC5653670 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples.

Roopnarain Ashira A Mukhuba Mashudu M Adeleke Rasheed R Moeletsi Mokhele M

3 Biotech 20171023 6

The anaerobic digestion (AD) of organic waste for biogas production has received much attention in recent years due to the increasing need for renewable energy and environmentally friendly waste management systems. Identification of the microbial community involved in AD aids in better understanding and optimising of the process. The choice of DNA extraction method is an integral step in any molecular biodiversity study. In the present study, potential biases introduced by DNA extraction methods ...[more]

PMID: 29071172

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Project description:BackgroundPrevious studies have demonstrated the importance of DNA extraction methods for molecular detection of Staphylococcus, an important bacterial group in cystic fibrosis (CF). We sought to evaluate the effect of enzymatic digestion (EnzD) prior to DNA extraction on bacterial communities identified in sputum and oropharyngeal swab (OP) samples from patients with CF.MethodsDNA from 81 samples (39 sputum and 42 OP) collected from 63 patients with CF was extracted in duplicate with and without EnzD. Bacterial communities were determined by rRNA gene sequencing, and measures of alpha and beta diversity were calculated. Principal Coordinate Analysis (PCoA) was used to assess differences at the community level and Wilcoxon Signed Rank tests were used to compare relative abundance (RA) of individual genera for paired samples with and without EnzD.ResultsShannon Diversity Index (alpha-diversity) decreased in sputum and OP samples with the use of EnzD. Larger shifts in community composition were observed for OP samples (beta-diversity, measured by Morisita-Horn), whereas less change in communities was observed for sputum samples. The use of EnzD with OP swabs resulted in significant increase in RA for the genera Gemella (p < 0.01), Streptococcus (p < 0.01), and Rothia (p < 0.01). Staphylococcus (p < 0.01) was the only genus with a significant increase in RA from sputum, whereas the following genera decreased in RA with EnzD: Veillonella (p < 0.01), Granulicatella (p < 0.01), Prevotella (p < 0.01), and Gemella (p = 0.02). In OP samples, higher RA of Gram-positive taxa was associated with larger changes in microbial community composition.DiscussionWe show that the application of EnzD to CF airway samples, particularly OP swabs, results in differences in microbial communities detected by sequencing. Use of EnzD can result in large changes in bacterial community composition, and is particularly useful for detection of Staphylococcus in CF OP samples. The enhanced identification of Staphylococcus aureus is a strong indication to utilize EnzD in studies that use OP swabs to monitor CF airway communities.

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Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples.

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Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples.

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