Laboratory contamination over time during low-biomass sample analysis

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (<i>e.g.</i> skin swabs, tissue biopsies, ice, water, degraded forensic samples, or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high-throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no-template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over five years to characterize long-term contaminant diversity. We additionally compared the contaminant content within a homemade silica-based extraction method, commonly used to analyse low-endogenous samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to the modern molecular biology laboratories, and changed over time according to researchers, month, and season. The commercial kit contained higher microbial diversity and several human-associated taxa in comparison to the homemade silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low-biomass metagenomic studies: 1) extraction blank controls should be included and sequenced with every batch of extractions and 2) the contributions of laboratory contamination should be assessed and reported in each high-throughput metagenomic study.<b> </b>