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.

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

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Background Aboriginal Australians and Torres Strait Islanders (hereafter respectfully referred to as Indigenous Australians) experience disproportionately poor health and low life expectancy compared to non-Indigenous Australians. Poor oral health is a critical, but understudied, contributor to this health gap. A considerable body of evidence links poor oral health to increased risks of other chronic non-communicable conditions, such as diabetes, cardiovascular disease, chronic kidney disease, and poor emotional wellbeing.  Main The oral microbiota is indisputably associated with several oral diseases that disproportionately affect Indigenous Australians. Furthermore, a growing literature suggests direct and indirect links between the oral microbiota and systemic chronic non-communicable diseases that underpin much of the Indigenous health gap in Australia. Recent research indicates that oral microbial communities are shaped by a combination of cultural and lifestyle factors and are inherited from caregivers to children. Systematic differences in oral microbiota diversity and composition have been identified between Indigenous and non-Indigenous individuals in Australia and elsewhere, suggesting that microbiota-related diseases may be distinct in Indigenous Australians.  Conclusion Oral microbiota research involving Indigenous Australians is a promising new area that could benefit Indigenous communities in numerous ways. These potential benefits include: (1) ensuring equity and access for Indigenous Australians in microbiota-related therapies; (2) opportunities for knowledge-sharing and collaborative research between scientists and Indigenous communities; and (3) using knowledge about the oral microbiota and chronic disease to help close the gaps in Indigenous oral and systemic health.

Australian Aboriginal and Torres Strait Islander children experience unacceptably high rates of dental caries compared to their non-Indigenous Australian counterparts. Dental caries significantly impacts the quality of life of children and their families, particularly in remote communities. While many socioeconomic and lifestyle factors impact caries risk, the central role of the oral microbiota in mediating dental caries has not been extensively investigated in these communities. Here, we examine factors that shape diversity and composition of the salivary microbiota in Aboriginal and Torres Strait Islander children and adolescents living in the remote Northern Peninsula Area (NPA) of Far North Queensland. We employed 16S ribosomal RNA amplicon sequencing to profile bacteria present in saliva collected from 205 individuals aged 4–17 years from the NPA. Higher average microbial diversity was generally linked to increased age and salivary pH, less frequent toothbrushing, and proxies for lower socioeconomic status (SES). Differences in microbial composition were significantly related to age, salivary pH, SES proxies, and active dental caries. Notably, a feature classified as Streptococcus sobrinus increased in abundance in children who reported less frequent tooth brushing. A specific Veillonella feature was associated with caries presence, while features classified as Actinobacillus/Haemophilus and Leptotrichia were associated with absence of caries; a Lactobacillus gasseri feature increased in abundance in severe caries. Finally, we statistically assessed the interplay between dental caries and caries risk factors in shaping the oral microbiota. These data provide a detailed understanding of biological, behavioral, and socioeconomic factors that shape the oral microbiota and may underpin caries development in this group. This information can be used in the future to improve tailored caries prevention and management options for Australian Aboriginal and Torres Strait Islander children and communities.

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Background and objectives Aboriginal Australians and Torres Strait Islanders (hereafter respectfully referred to as Indigenous Australians) experience a high burden of chronic non-communicable diseases (NCDs). Increased NCD risk is linked to oral diseases mediated by the oral microbiota, a microbial community influenced by both vertical transmission and lifestyle factors. As an initial step towards understanding the oral microbiota as a factor in Indigenous health, we present the first investigation of oral microbiota in Indigenous Australian adults. Methodology Dental calculus samples from Indigenous Australians with periodontal disease (PD) (n = 13) and non-Indigenous individuals both with (n = 18) and without PD (n = 20) were characterized using 16S rRNA gene amplicon sequencing. Alpha and beta diversity, differentially abundant microbial taxa, and taxa unique to different participant groups were analysed using QIIME2. Results Samples from Indigenous Australians were more phylogenetically diverse (Kruskal-Wallis H = 19.86, p = 8.3 x 1 0 −6), differed significantly in composition from non-Indigenous samples (PERMANOVA pseudo-F = 10.42, p = 0.001), and contained a relatively high proportion of unique taxa not previously reported in the human oral microbiota (e.g. Endomicrobia). These patterns were robust to stratification by PD status. Oral microbiota diversity and composition also differed between Indigenous individuals living in different geographic regions. Conclusions and implications Indigenous Australians may harbour unique oral microbiota shaped by their long relationships with Country (ancestral homelands). Our findings have implications for understanding the origins of oral and systemic NCDs and for inclusion of Indigenous peoples in microbiota research, highlighting the microbiota as a novel field of enquiry to improve Indigenous health. Lay Summary The community of microorganisms in the mouth (oral microbiota) has recently been linked to several chronic diseases that disproportionately impact Indigenous Australians. In this study, oral microbiota differ significantly between Indigenous Australians and non-Indigenous counterparts, suggesting the microbiota could be a novel factor with potential to improve Indigenous health outcomes.