A need for standardization in drinking water analysis – an investigation of DNA extraction procedure, primer choice and detection limit of 16S rRNA amplicon sequencing

Today 16S rRNA amplicon sequencing is a widely utilized technique for analyzing the bacterial community structure in drinking water. Concurrently with the prevalence of this method, the biases associated with 16S rRNA amplicon sequencing have been well-documented. However, no comprehensive attempts have been made to illuminate the effects specifically related to bacterial communities in drinking water. In this study, we investigated the impact of the DNA extraction and primer choice on the observed community structure, and we also estimated the detection limit of the 16S rRNA amplicon sequencing. The PowerWater DNA Isolation Kit resulted in significantly higher amounts of isolated DNA compared to the FastDNA SPIN Kit for Soil. Furthermore, extraction with the PowerWater kit lead to detection of a significantly higher number of OTUs. Likewise, the primer experiment revealed great discrepancies in OTU abundances between primers targeting the V1-3, V3-4 and V4 region of the 16S rRNA gene. Some of the primers proved unable to detect entire phyla. Estimations of the detection limit were based on bacteria free water samples (~1 L) spiked with E. coli cells in different concentrations [101 – 106 cells/mL]. E. coli could be detected in all samples. However, samples with 101 cells/mL had several contaminating OTUs, constituting approximately 8% of the read abundances. For 16S rRNA gene analysis in drinking water samples, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination with PCR-amplification of the V3-4 region.