Profiling soil microbial communities with next-generation sequencing: The influence of DNA kit selection and technician technical expertise

Abstract

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of nextgeneration sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1)MO BIO PowerSoil ® DNA Isolation kit (MO_R andMO_T) and (2) NucleoSpin ® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P < 0:006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.