American Gut: an Open Platform for Citizen Science Microbiome Research

McDonald D, Hyde E, Debelius JW, et al. American Gut: an Open Platform for Citizen Science Microbiome Research. mSystems. 2018;3(3):e00031-18

Drool rules! Novel methods to further knowledge of microbial communities in the human oral cavity

As I am preparing my dissertation, a global Coronavirus pandemic is spreading, affecting the way we travel, work, and communicate. Transmitted primarily via respiratory droplets, this virus spread across the entire globe in a matter of months, highlighting the interconnectedness of modern life and the microscopic world which invisibly underlies all our interactions.In fact the vast majority of life on earth is microbial, and for the vast majority of time microorganisms were the only life present. Animals emerged in a robust microbial world, and our evolution was shaped by and from these microorganisms.Even during this global pandemic, it is easy to view ourselves as distinct, independent organisms. But recent evidence challenges this notion. Roughly 1% of our body weight is comprised of trillions of microorganisms, collectively referred to as the human microbiome. Our microbiomes are necessary for healthy nutrition, immune function, and even reproduction. How our microbiomes are formed, maintained, shared, and perturbed are active areas of research. My doctoral research focused on understanding and improving the molecular tools used to evaluate microbial communities and applying those tools to better understand human microbiomes.This dissertation begins with three reviews summarizing what we know about human microbiomes and what is still lacking. Chapters 2 and 3 describe benchtop and computational advances to the human microbiome field, respectively. The final chapter includes two research articles currently under review describing the application of these tools to gain novel insight about human oral microorganisms.Throughout my research, I found that human saliva served as a valuable tool for developing these novel techniques because it is simple to collect, high biomass, and relatively easy to manipulate. So although it was not my original intention, I have come to appreciate saliva as a valuable resource for pushing the boundary of human microbiome research. It is my hope that this research improves our ability to evaluate microbial communities, allowing for future research on altering microbiomes to improve human and environmental health

Improving saliva shotgun metagenomics by chemical host DNA depletion.

BackgroundShotgun sequencing of microbial communities provides in-depth knowledge of the microbiome by cataloging bacterial, fungal, and viral gene content within a sample, providing an advantage over amplicon sequencing approaches that assess taxonomy but not function and are taxonomically limited. However, mammalian DNA can dominate host-derived samples, obscuring changes in microbial populations because few DNA sequence reads are from the microbial component. We developed and optimized a novel method for enriching microbial DNA from human oral samples and compared its efficiency and potential taxonomic bias with commercially available kits.ResultsThree commercially available host depletion kits were directly compared with size filtration and a novel method involving osmotic lysis and treatment with propidium monoazide (lyPMA) in human saliva samples. We evaluated the percentage of shotgun metagenomic sequencing reads aligning to the human genome, and taxonomic biases of those not aligning, compared to untreated samples. lyPMA was the most efficient method of removing host-derived sequencing reads compared to untreated sample (8.53 ± 0.10% versus 89.29 ± 0.03%). Furthermore, lyPMA-treated samples exhibit the lowest taxonomic bias compared to untreated samples.ConclusionOsmotic lysis followed by PMA treatment is a cost-effective, rapid, and robust method for enriching microbial sequence data in shotgun metagenomics from fresh and frozen saliva samples and may be extensible to other host-derived sample types

Improving saliva shotgun metagenomics by chemical host DNA depletion

Abstract Background Shotgun sequencing of microbial communities provides in-depth knowledge of the microbiome by cataloging bacterial, fungal, and viral gene content within a sample, providing an advantage over amplicon sequencing approaches that assess taxonomy but not function and are taxonomically limited. However, mammalian DNA can dominate host-derived samples, obscuring changes in microbial populations because few DNA sequence reads are from the microbial component. We developed and optimized a novel method for enriching microbial DNA from human oral samples and compared its efficiency and potential taxonomic bias with commercially available kits. Results Three commercially available host depletion kits were directly compared with size filtration and a novel method involving osmotic lysis and treatment with propidium monoazide (lyPMA) in human saliva samples. We evaluated the percentage of shotgun metagenomic sequencing reads aligning to the human genome, and taxonomic biases of those not aligning, compared to untreated samples. lyPMA was the most efficient method of removing host-derived sequencing reads compared to untreated sample (8.53 ± 0.10% versus 89.29 ± 0.03%). Furthermore, lyPMA-treated samples exhibit the lowest taxonomic bias compared to untreated samples. Conclusion Osmotic lysis followed by PMA treatment is a cost-effective, rapid, and robust method for enriching microbial sequence data in shotgun metagenomics from fresh and frozen saliva samples and may be extensible to other host-derived sample types

Improving saliva shotgun metagenomics by chemical host DNA depletion.

BackgroundShotgun sequencing of microbial communities provides in-depth knowledge of the microbiome by cataloging bacterial, fungal, and viral gene content within a sample, providing an advantage over amplicon sequencing approaches that assess taxonomy but not function and are taxonomically limited. However, mammalian DNA can dominate host-derived samples, obscuring changes in microbial populations because few DNA sequence reads are from the microbial component. We developed and optimized a novel method for enriching microbial DNA from human oral samples and compared its efficiency and potential taxonomic bias with commercially available kits.ResultsThree commercially available host depletion kits were directly compared with size filtration and a novel method involving osmotic lysis and treatment with propidium monoazide (lyPMA) in human saliva samples. We evaluated the percentage of shotgun metagenomic sequencing reads aligning to the human genome, and taxonomic biases of those not aligning, compared to untreated samples. lyPMA was the most efficient method of removing host-derived sequencing reads compared to untreated sample (8.53 ± 0.10% versus 89.29 ± 0.03%). Furthermore, lyPMA-treated samples exhibit the lowest taxonomic bias compared to untreated samples.ConclusionOsmotic lysis followed by PMA treatment is a cost-effective, rapid, and robust method for enriching microbial sequence data in shotgun metagenomics from fresh and frozen saliva samples and may be extensible to other host-derived sample types

A Novel Sparse Compositional Technique Reveals Microbial Perturbations

By accounting for the sparse compositional nature of microbiome data sets, robust Aitchison PCA can yield high discriminatory power and salient feature ranking between microbial niches. The software to perform this analysis is available under an open-source license and can be obtained at https://github.com/biocore/DEICODE; additionally, a QIIME 2 plugin is provided to perform this analysis at https://library.qiime2.org/plugins/q2-deicode.The central aims of many host or environmental microbiome studies are to elucidate factors associated with microbial community compositions and to relate microbial features to outcomes. However, these aims are often complicated by difficulties stemming from high-dimensionality, non-normality, sparsity, and the compositional nature of microbiome data sets. A key tool in microbiome analysis is beta diversity, defined by the distances between microbial samples. Many different distance metrics have been proposed, all with varying discriminatory power on data with differing characteristics. Here, we propose a compositional beta diversity metric rooted in a centered log-ratio transformation and matrix completion called robust Aitchison PCA. We demonstrate the benefits of compositional transformations upstream of beta diversity calculations through simulations. Additionally, we demonstrate improved effect size, classification accuracy, and robustness to sequencing depth over the current methods on several decreased sample subsets of real microbiome data sets. Finally, we highlight the ability of this new beta diversity metric to retain the feature loadings linked to sample ordinations revealing salient intercommunity niche feature importance

Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary

Conventional wisdom holds that PCR amplification for sequencing should employ pooled replicate reactions to reduce bias due to jackpot effects and chimera formation. However, modern amplicon data analysis employs methods that may be less sensitive to such artifacts. Here we directly compare results from single versus triplicate reactions for 16S amplicon sequencing and find no significant impact of adopting a less labor-intensive single-reaction protocol

Antibiotic-induced microbiome depletion alters metabolic homeostasis by affecting gut signaling and colonic metabolism

Antibiotic-induced microbiome depletion is one of the most common approaches to modulate the gut microbiome. Here the authors demonstrate that it affects gut homeostasis and glucose metabolism by decreasing luminal short chain fatty acids and leading to a shift of energy utilization by colonocytes