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Back to Basics – The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities

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  • Mads Albertsen
  • Søren M Karst
  • Anja S Ziegler
  • Rasmus H Kirkegaard
  • Per H Nielsen

Abstract

DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers.

Suggested Citation

  • Mads Albertsen & Søren M Karst & Anja S Ziegler & Rasmus H Kirkegaard & Per H Nielsen, 2015. "Back to Basics – The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities," PLOS ONE, Public Library of Science, vol. 10(7), pages 1-15, July.
    • Handle: RePEc:plo:pone00:0132783
    DOI: 10.1371/journal.pone.0132783
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    References listed on IDEAS

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    1. Wickham, Hadley, 2007. "Reshaping Data with the reshape Package," Journal of Statistical Software, Foundation for Open Access Statistics, vol. 21(i12).
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    2. Amanda Sörensen Ristinmaa & Albert Tafur Rangel & Alexander Idström & Sebastian Valenzuela & Eduard J. Kerkhoven & Phillip B. Pope & Merima Hasani & Johan Larsbrink, 2023. "Resin acids play key roles in shaping microbial communities during degradation of spruce bark," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
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    4. Thomas Bygh Nymann Jensen & Sebastian Mølvang Dall & Simon Knutsson & Søren Michael Karst & Mads Albertsen, 2024. "High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing," PLOS ONE, Public Library of Science, vol. 19(4), pages 1-16, April.
    5. Sposób, Michał, 2024. "Optimization of ex-situ biomethanation process in trickle bed reactor: The impact of slight H2/CO2 ratio adjustments and different packing materials," Renewable Energy, Elsevier, vol. 222(C).

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