Variability in the pre-analytical stages influences microbiome laboratory analyses
Authors
Kumar, AditiGravdal, Kristin
Segal, Jonathan P
Steed, Helen

Brookes, Matthew

Al-Hassi, Hafid Omar
Issue Date
2022-06-15
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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.Citation
Kumar A, Gravdal K, Segal JP, Steed H, Brookes MJ, Al-Hassi HO. (2022) Variability in the Pre-Analytical Stages Influences Microbiome Laboratory Analyses. Genes, 13(6):1069. https://doi.org/10.3390/genes13061069Publisher
MDPIJournal
GenesPubMed ID
35741831 (pubmed)Additional Links
https://www.mdpi.com/2073-4425/13/6/1069Type
Journal articleLanguage
enDescription
© 2022 The Authors. Published by MDPI. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.3390/genes13061069ISSN
2073-4425EISSN
2073-4425Sponsors
This study was supported by the Research Investment Fund-4 (RIF4) from the University of Wolverhampton, Wolverhampton WV1 1LY UK.ae974a485f413a2113503eed53cd6c53
10.3390/genes13061069
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Except where otherwise noted, this item's license is described as Licence for published version: Creative Commons Attribution 4.0 International
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