Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study

Publication date: Available online 11 July 2019Source: Journal of Microbiological MethodsAuthor(s): Alan C. Ezzy, Amanda D. Hagstrom, Chris George, Adam S. Hamlin, Lily Pereg, Aron J. Murphy, Gal WinterAbstractHuman gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7–164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at −80 °C as a control treatment. DNA yield and purity was ...
Source: Journal of Microbiological Methods - Category: Microbiology Source Type: research