No abstract

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 (e.g., 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 5 years to characterize long‐term contaminant diversity. We additionally compared the contaminant content within a home‐made silica‐based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human‐associated taxa in comparison to the home‐made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low‐biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high‐throughput metagenomic study.

Ancient DNA (aDNA) encompasses nonmodern genetic material (i.e., from dead organisms), which is typically highly fragmented and damaged (Shapiro & Hofreiter, 2012). Analyses of aDNA in marine sediments have become increasingly common (e.g., Coolen

17Bacteria are not only ubiquitous on earth but can also be incredibly diverse 18 within clean laboratories and reagents. The presence of both living and dead bacteria 19 in laboratory environments and reagents is especially problematic when examining 20 samples with low endogenous content (e.g. skin swabs, tissue biopsies, ice, water, 21 degraded forensic samples, or ancient material), where contaminants can outnumber 22 endogenous microorganisms within samples. The contribution of contaminants within 23 high-throughput studies remains poorly understood because of the relatively low 24 number of contaminant surveys. Here, we examined 144 negative control samples 25 (extraction blank and no-template amplification controls) collected in both typical 26 molecular laboratories and an ultraclean ancient DNA laboratory over five years to 27 characterize long-term contaminant diversity. We additionally compared the 28 contaminant content within a homemade silica-based extraction method, commonly 29 used to analyse low-endogenous samples, with a widely used commercial DNA 30 extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA 31 laboratory was unique compared to the modern molecular biology laboratories, and 32 changed over time according to researchers, month, and season. The commercial kit 33 contained higher microbial diversity and several human-associated taxa in comparison 34to the homemade silica extraction protocol. We recommend a minimum of two 35 strategies to reduce the impacts of laboratory contaminants within low-biomass 36 metagenomic studies: 1) extraction blank controls should be included and sequenced 37 with every batch of extractions and 2) the contributions of laboratory contamination 38 should be assessed and reported in each high-throughput metagenomic study. 39 3 Main Text: 40In the new era of culture-independent microbiome research, targeted amplicon 41 or 'metabarcoding' approaches are now routinely used to amplify DNA from 42 microbial species across the tree of life. However, these methods lack the ability to 43 select for either specific species or to exclude contaminants [1]. Although these 44 techniques have provided invaluable insight into otherwise cryptic microbial 45 communities, the increased sensitivity and lack of target specificity leaves microbiota 46 studies particularly susceptible to the effects of contamination. Such effects are 47 widespread, as several recent studies have indicated that contaminant microbial DNA 48 can be routinely isolated from laboratory reagents and surfaces [2][3][4] and that this 49 signal has significantly impacted the interpretation and characterization of microbiota 50 in high-throughput sequencing studies. For example, Salter et al. recently 51 demonstrated that bacterial DNA present in laboratory reagents is present in both 52 quality-filtered 16S ribosomal RNA (rRNA) gene and shotgun metagenomic datasets 53 and significantly impacts the interpretation of results [3]. Multiple microbial 54