Dataset Information

Laboratory contamination over time during low-biomass sample analysis.

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.

SUBMITTER: Weyrich LS

PROVIDER: S-EPMC6850301 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Laboratory contamination over time during low-biomass sample analysis.

Weyrich Laura S LS Farrer Andrew G AG Eisenhofer Raphael R Arriola Luis A LA Young Jennifer J Selway Caitlin A CA Handsley-Davis Matilda M Adler Christina J CJ Breen James J Cooper Alan A

Molecular ecology resources 20190429 4

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-thro ...[more]

PMID: 30887686

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Project description:Background The blanket usage of antimicrobials at the end of lactation (or “drying off”) in dairy cattle is under increasing scrutiny due to concerns about antimicrobial resistance. To lower antimicrobial usage in dairy farming, farmers are now encouraged to use “selective dry cow therapy” whereby only cows viewed as at high risk of mastitis are administered antimicrobial agents. It is important to gain a better understanding of how this practice affects the udder-associated microbiota and the potential knock-on effects on antimicrobial-resistant bacterial populations circulating on the farm. However, there are challenges associated with studying low biomass environments such as milk, due to known contamination effects on microbiome datasets. Here, we obtained milk samples from cattle at drying off and at calving to measure potential shifts in bacterial load and microbiota composition, with a critical assessment of contamination effects. Results Several samples had no detectable 16S rRNA gene copies and crucially, exogenous contamination was detected in the initial microbiome dataset. The affected samples were removed from the final microbiome analysis, which compromised the experimental design and statistical analysis. There was no significant difference in bacterial load between treatments (P > 0.05), but load was lower at calving than at drying off (P = 0.039). Escherichia coli counts by both sequence and culture data increased significantly in the presence of reduced bacterial load and a decreasing trend of microbiome richness and diversity. The milk samples revealed diverse microbiomes not reflecting a typical infection profile and were largely comprised of gut- and skin-associated taxa, with the former decreasing somewhat after prolonged sealing of the teats. Conclusions The drying off period had a key influence on microbiota composition and bacterial load, which appeared to be independent of antimicrobial usage. The interactions between drying off treatment protocol and milk microbiome dynamics are clearly complex, and our evaluations of these interactions were restricted by low biomass samples and contamination effects. Therefore, our analysis will inform the design of future studies to establish whether different selection protocols could be implemented to further minimise antimicrobial usage. Supplementary Information The online version contains supplementary material available at 10.1186/s42523-021-00144-x.

Project description:The major aquatic interface between host and environment in teleost finfish species is the gill. The diversity of this infraclass, high complexity of the organ, and its direct exposure to the surrounding environment make it an ideal candidate for furthering our understanding of the intertwined relationships between host and microbiome. Capturing the structure and diversity of bacterial communities from this low-biomass, inhibitor-rich tissue can, however, prove challenging. Lessons learned in doing so are directly applicable to similar sample types in other areas of microbiology. Through the development of a quantitative PCR assay for both host material and 16S rRNA genes, we tested and developed a robust method for low-biomass sample collection which minimized host DNA contamination. Quantification of 16S rRNA facilitated not only the screening of samples prior to costly library construction and sequencing but also the production of equicopy libraries based on 16S rRNA gene copies. A significant increase in diversity of bacteria captured was achieved, providing greater information on the true structure of the microbial community. Such findings offer important information for determining functional processes. Results were confirmed across fresh, brackish, and marine environs with four different fish species, with results showing broad homology between samples, demonstrating the robustness of the approach. Evidence presented is widely applicable to samples similar in composition, such as sputum or mucus, or those that are challenging due to the inherent inclusion of inhibitors. IMPORTANCE The interaction between the fish gill and surrounding bacteria-rich water provides an intriguing model for examining the interaction between the fish, free-floating bacteria, and the bacterial microbiome on the gill surface. Samples that are inherently low in bacteria, or that have components that inhibit the ability to produce libraries that identify the components of microbial communities, present significant challenges. Gill samples present both of these types of challenges. We developed methods for quantifying both the bacterial and host DNA material and established a sampling method which both reduced inhibitor content and maximized bacterial diversity. By quantifying and normalizing bacteria prior to library construction, we showed significant improvements with regards to the fidelity of the final data. Our results support wide-ranging applications for analyzing samples of similar composition, such as mucus and sputum, in other microbiological spheres.

Dataset Information

Laboratory contamination over time during low-biomass sample analysis.

Publications

Laboratory contamination over time during low-biomass sample analysis.

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