Abstract
Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.
Highlights
The gut microbiome plays a key role in shaping human health and has been the subject of an increasing number of studies in the context of disease development, diagnostics and treatment
Each volunteer collected the samples from the same stool sample using three different sampling kits (SK): a stool container (SK1); a flocked swab (SK2) and a cotton swab (SK3)
In an attempt to elucidate some of the factors determining the success of such studies, we focused on the effects of sampling and DNA extraction methods on a number of relevant variables from DNA integrity to final bacterial composition at different taxa levels
Summary
The gut microbiome plays a key role in shaping human health and has been the subject of an increasing number of studies in the context of disease development, diagnostics and treatment. Www.nature.com/scientificreports targeting different parts of the 16S rRNA gene[40,41] and data analysis[42] All of these factors may lead to the misinterpretation of changes in the microbiome and hamper direct comparisons of results between individual studies[43,44,45]. The combination of enzymatic and mechanical disruption is recommended as more effective in the lysis of Gram-positive bacteria[8,22,26,34,35,37,39] These DNA extraction comparison studies are limited to a rather small number of individuals (from 2 to 9) and none of them compared the kits in terms of DNA yield and quality, presence of PCR inhibitors, the human to bacterial DNA ratio, the efficiency of Gram-positive bacteria cell wall lysis and the observed bacterial composition at different taxa levels all at once. The aim of our study was to perform systematic assessment of effect of sampling and DNA isolation kits and their combinations on a full range of parameters of bacterial DNA quality, bacterial diversity and composition, with respect to user acceptance
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