Stool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform
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Date
2019-09-25
Authors
Vídeňská, Petra
Šmerková, Kristýna
Zwinsová, Barbora
Popovici, Vlad
Micenková, Lenka
Sedlář, Karel
Budinská, Eva
ORCID
0000-0002-8269-4020Advisor
Referee
Mark
Journal Title
Journal ISSN
Volume Title
Publisher
Springer Nature
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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.
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Citation
Scientific Reports. 2019, vol. 2019, issue 9, p. 1-14.
https://www.nature.com/articles/s41598-019-49520-3
https://www.nature.com/articles/s41598-019-49520-3
Document type
Peer-reviewed
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Published version
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Language of document
en