The impact of sampling, PCR, and sequencing replication on discerning changes in drinking water bacterial community over diurnal time-scales.

High-throughput and deep DNA sequencing, particularly amplicon sequencing, is being increasingly utilized to reveal spatial and temporal dynamics of bacterial communities in drinking water systems. Whilst the sampling and methodological biases associated with PCR and sequencing have been studied in other environments, they have not been quantified for drinking water. These biases are likely to have the greatest effect on the ability to characterize subtle spatio-temporal patterns influenced by process/environmental conditions. In such cases, intra-sample variability may swamp any underlying small, systematic variation. To evaluate this, we undertook a study with replication at multiple levels including sampling sites, sample collection, PCR amplification, and high throughput sequencing of 16S rRNA amplicons. The variability inherent to the PCR amplification and sequencing steps is significant enough to mask differences between bacterial communities from replicate samples. This was largely driven by greater variability in detection of rare bacteria (relative abundance <0.01%) across PCR/sequencing replicates as compared to replicate samples. Despite this, we captured significant changes in bacterial community over diurnal time-scales and find that the extent and pattern of diurnal changes is specific to each sampling location. Further, we find diurnal changes in bacterial community arise due to differences in the presence/absence of the low abundance bacteria and changes in the relative abundance of dominant bacteria. Finally, we show that bacterial community composition is significantly different across sampling sites for time-periods during which there are typically rapid changes in water use. This suggests hydraulic changes (driven by changes in water demand) contribute to shaping the bacterial community in bulk drinking water over diurnal time-scales.