Variability in the Pre-Analytical Stages Influences Microbiome Laboratory Analyses

Genes (Basel). 2022 Jun 15;13(6):1069. doi: 10.3390/genes13061069.


Introduction: There are numerous confounding variables in the pre-analytical steps in the analysis of gut microbial composition that affect data consistency and reproducibility. This study compared two DNA extraction methods from the same faecal samples to analyse differences in microbial composition. Methods: DNA was extracted from 20 faecal samples using either (A) chemical/enzymatic heat lysis (lysis buffer, proteinase K, 95 °C + 70 °C) or (B) mechanical and chemical/enzymatic heat lysis (bead-beating, lysis buffer, proteinase K, 65 °C). Gut microbiota was mapped through the 16S rRNA gene (V3−V9) using a set of pre-selected DNA probes targeting >300 bacteria on different taxonomic levels. Apart from the pre-analytical DNA extraction technique, all other parameters including microbial analysis remained the same. Bacterial abundance and deviations in the microbiome were compared between the two methods. Results: Significant variation in bacterial abundance was seen between the different DNA extraction techniques, with a higher yield of species noted in the combined mechanical and heat lysis technique (B). The five predominant bacteria seen in both (A) and (B) were Bacteroidota spp. and Prevotella spp. (p = NS), followed by Bacillota (p = 0.005), Lachhnospiraceae (p = 0.0001), Veillonella spp. (p < 0.0001) and Clostridioides (p < 0.0001). Conclusion: As microbial testing becomes more easily and commercially accessible, a unified international consensus for optimal sampling and DNA isolation procedures must be implemented for robustness and reproducibility of the results.

Keywords: 16S rRNA; DNA; microbiome; pre-analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / genetics
  • DNA
  • DNA, Bacterial / analysis
  • DNA, Bacterial / genetics
  • Endopeptidase K
  • Microbiota* / genetics
  • RNA, Ribosomal, 16S / genetics
  • Reproducibility of Results


  • DNA, Bacterial
  • RNA, Ribosomal, 16S
  • DNA
  • Endopeptidase K

Grants and funding

This study was supported by the Research Investment Fund-4 (RIF4) from the University of Wolverhampton, Wolverhampton WV1 1LY UK.