Evaluating Methods for the Optimal Extraction of Microbial Nucleic Acids for Analysis of the Human Gut Microbiota

Abstract

The human gut contains dense and diverse microbial communities which have profound influences on human health. Several diseases and disorders, including Autism Spectrum Disorder, have been linked to dysbiosis (imbalance) in these communities. Gaining meaningful insights into the gut microbial community requires provision of high quality microbial nucleic acids from human faecal samples, however a consensus regarding microbial DNA extraction methods that capture true gut community diversity has not yet been reached. This thesis presents a systematic analysis of commonly used microbial DNA extraction methods, and has identified significant sources of variation, as well as their impact on community diversity estimates. Furthermore, this is the first methods comparison study to independently analyse the extraction method used by the Human Microbiome Project. Five commonly used DNA extraction methods (Human Microbiome Project protocol, MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Stool Mini Kit, ZR Fecal DNA MiniPrep™, and one non-kit phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality (A260/A280 ratio), integrity, and microbial community structure based on sequencing of the amplified V4 region of the bacterial and archaeal 16S rRNA gene. Three neurotypical female subjects in their mid-20s, with no dietary restrictions or recent antibiotic usage (>6 months), and located within Auckland, New Zealand, consented to supply three stool samples each. The results indicate that the largest portion of variation within the model was attributed to differences between subjects, with a smaller proportion of variation due to DNA extraction method. Comparisons between methods suggest the most effective microbial DNA extraction method is the MoBio PowerSoil® DNA Isolation Kit. The consistently high yield and quality of extracted DNA, as well as minimal shearing, from this kit support this decision. The microbial community profile of the ZR Fecal DNA MiniPrep™ kit was significantly different from that of the other methods; however, the other four methods recovered similar microbial communities. The findings of this research will be applied to a larger study investigating the role of the gut microbiota in Autism Spectrum Disorder in New Zealand, conducted under the auspices of “Minds for Minds”.