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

PLoS One. 2015 Jul 16;10(7):e0132783. doi: 10.1371/journal.pone.0132783. eCollection 2015.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers / genetics
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / isolation & purification
  • Gram-Negative Bacteria / classification
  • Gram-Negative Bacteria / genetics*
  • Gram-Positive Bacteria / classification
  • Gram-Positive Bacteria / genetics*
  • In Situ Hybridization, Fluorescence
  • Metagenomics
  • Microbial Consortia / genetics
  • Phylogeny*
  • Polymerase Chain Reaction / methods
  • RNA, Ribosomal, 16S / genetics
  • Sequence Analysis, DNA
  • Sewage / microbiology*
  • Solid Phase Extraction / methods*


  • DNA Primers
  • DNA, Bacterial
  • RNA, Ribosomal, 16S
  • Sewage

Grant support

This study was funded by the Innovation Fond Denmark (EcoDesign-MBR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.