Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia-A Microbiome and Gene Profiling Study and Focused Review.

Oral potentially malignant disorders (OPMDs) are a group of conditions that carry a risk of oral squamous cell carcinoma (OSCC) development. Recent studies indicate that periodontal disease-associated pathogenic bacteria may play a role in the transition from healthy mucosa to dysplasia and to OSCC. Yet, the microbial signatures associated with the transition from healthy mucosa to dysplasia have not been established. To characterize oral microbial signatures at these different sites, we performed a 16S sequencing analysis of both oral swab and formalin-fixed, paraffin-embedded tissue (FFPE) samples. We collected oral swabs from healthy mucosa (from healthy patients), histologically normal mucosa adjacent to dysplasia, and low-grade oral dysplasia. Additionally, FFPE samples from histologically normal mucosa adjacent to OSCC, plus low grade and high-grade oral dysplasia samples were also collected. The collected data demonstrate significant differences in the alpha and beta microbial diversities of different sites in oral mucosa, dysplasia, and OSCC, as well as increased dissimilarities within these sites. We found that the Proteobacteria phyla abundance increased, concurrent with a progressive decrease in the Firmicutes phyla abundance, as well as altered levels of Enterococcus cecorum, Fusobacterium periodonticum, Prevotella melaninogenica, and Fusobacterium canifelinum when moving from healthy to diseased sites. Moreover, the swab sample analysis indicates that the oral microbiome may be altered in areas that are histologically normal, including in mucosa adjacent to dysplasia. Furthermore, trends in specific microbiome changes in oral swab samples preceded those in the tissues, signifying early detection opportunities for clinical diagnosis. In addition, we evaluated the gene expression profile of OSCC cells (HSC-3) infected with either P. gingivalis, T. denticola, F. nucelatum, or S. sanguinis and found that the three periodontopathogens enrich genetic processes related to cancer progression, including skin keratinization/cornification, while the commensal enriched processes related to RNA processing and adhesion. Finally, we reviewed the dysplasia microbiome literature and found a significant decrease in commensal bacteria, such as the Streptococci genus, and a simultaneous increase in pathogenic bacteria, mainly Bacteroidetes phyla and Fusobacterium genus. These findings suggest that features of the oral microbiome can serve as novel biomarkers for dysplasia and OSCC disease progression.

Understanding natural selection in Holocene Europe using multi-locus genotype identity scans.

Ancient DNA (aDNA) has been a revolutionary technology in understanding human history but has not been used extensively to study natural selection as large sample sizes to study allele frequency changes over time have thus far not been available. Here, we examined a time transect of 708 published samples over the past 7,000 years of European history using multi-locus genotype-based selection scans. As aDNA data is affected by high missingness, ascertainment bias, DNA damage, random allele calling, and is unphased, we first validated our selection scan, G12ancient, on simulated data resembling aDNA under a demographic model that captures broad features of the allele frequency spectrum of European genomes as well as positive controls that have been previously identified and functionally validated in modern European datasets on data from ancient individuals from time periods very close to the present time. We then applied our statistic to the aDNA time transect to detect and resolve the timing of natural selection occurring genome wide and found several candidates of selection across the different time periods that had not been picked up by selection scans using single SNP allele frequency approaches. In addition, enrichment analysis discovered multiple categories of complex traits that might be under adaptation across these periods. Our results demonstrate the utility of applying different types of selection scans to aDNA to uncover putative selection signals at loci in the ancient past that might have been masked in modern samples.

SNV-FEAST: microbial source tracking with single nucleotide variants.

Elucidating the sources of a microbiome can provide insight into the ecological dynamics responsible for the formation of these communities. Source tracking approaches to date leverage species abundance information; however, single nucleotide variants (SNVs) may be more informative because of their high specificity to certain sources. To overcome the computational burden of utilizing all SNVs for a given sample, we introduce a novel method to identify signature SNVs for source tracking. Signature SNVs used as input into a previously designed source tracking algorithm, FEAST, can more accurately estimate contributions than species and provide novel insights, demonstrated in three case studies.

Community diversity is associated with intra-species genetic diversity and gene loss in the human gut microbiome.

How the ecological process of community assembly interacts with intra-species diversity and evolutionary change is a longstanding question. Two contrasting hypotheses have been proposed: Diversity Begets Diversity (DBD), in which taxa tend to become more diverse in already diverse communities, and Ecological Controls (EC), in which higher community diversity impedes diversification. Previously, using 16S rRNA gene amplicon data across a range of microbiomes, we showed a generally positive relationship between taxa diversity and community diversity at higher taxonomic levels, consistent with the predictions of DBD (Madi et al., 2020). However, this positive 'diversity slope' plateaus at high levels of community diversity. Here we show that this general pattern holds at much finer genetic resolution, by analyzing intra-species strain and nucleotide variation in static and temporally sampled metagenomes from the human gut microbiome. Consistent with DBD, both intra-species polymorphism and strain number were positively correlated with community Shannon diversity. Shannon diversity is also predictive of increases in polymorphism over time scales up to ~4-6 months, after which the diversity slope flattens and becomes negative - consistent with DBD eventually giving way to EC. Finally, we show that higher community diversity predicts gene loss at a future time point. This observation is broadly consistent with the Black Queen Hypothesis, which posits that genes with functions provided by the community are less likely to be retained in a focal species' genome. Together, our results show that a mixture of DBD, EC, and Black Queen may operate simultaneously in the human gut microbiome, adding to a growing body of evidence that these eco-evolutionary processes are key drivers of biodiversity and ecosystem function.

Enrichment of Hard Sweeps on the X Chromosome in Drosophila melanogaster.

The characteristic properties of the X chromosome, such as male hemizygosity and its unique inheritance pattern, expose it to natural selection in a way that can be different from the autosomes. Here, we investigate the differences in the tempo and mode of adaptation on the X chromosome and autosomes in a population of Drosophila melanogaster. Specifically, we test the hypothesis that due to hemizygosity and a lower effective population size on the X, the relative proportion of hard sweeps, which are expected when adaptation is gradual, compared with soft sweeps, which are expected when adaptation is rapid, is greater on the X than on the autosomes. We quantify the incidence of hard versus soft sweeps in North American D. melanogaster population genomic data with haplotype homozygosity statistics and find an enrichment of the proportion of hard versus soft sweeps on the X chromosome compared with the autosomes, confirming predictions we make from simulations. Understanding these differences may enable a deeper understanding of how important phenotypes arise as well as the impact of fundamental evolutionary parameters on adaptation, such as dominance, sex-specific selection, and sex-biased demography.

Pervasive selective sweeps across human gut microbiomes.

The human gut microbiome is composed of a highly diverse consortia of species which are continually evolving within and across hosts. The ability to identify adaptations common to many host gut microbiomes would not only reveal shared selection pressures across hosts, but also key drivers of functional differentiation of the microbiome that may affect community structure and host traits. However, to date there has not been a systematic scan for adaptations that have spread across host microbiomes. Here, we develop a novel selection scan statistic, named the integrated linkage disequilibrium score (iLDS), that can detect the spread of adaptive haplotypes across host microbiomes via migration and horizontal gene transfer. Specifically, iLDS leverages signals of hitchhiking of deleterious variants with the beneficial variant, a common feature of adaptive evolution. We find that iLDS is capable of detecting simulated and known cases of selection, and moreover is robust to potential confounders that can also elevate LD. Application of the statistic to ~20 common commensal gut species from a large cohort of healthy, Western adults reveals pervasive spread of selected alleles across human microbiomes mediated by horizontal gene transfer. Among the candidate selective sweeps recovered by iLDS is an enrichment for genes involved in the metabolism of maltodextrin, a synthetic starch that has recently become a widespread component of Western diets. In summary, we demonstrate that selective sweeps across host microbiomes are a common feature of the evolution of the human gut microbiome.

Rapid evolution and strain turnover in the infant gut microbiome.

Although the ecological dynamics of the infant gut microbiome have been intensely studied, relatively little is known about evolutionary dynamics in the infant gut microbiome. Here we analyze longitudinal fecal metagenomic data from more than 700 infants and their mothers over the first year of life and find that the evolutionary dynamics in infant gut microbiomes are distinct from those of adults. We find evidence for more than a 10-fold increase in the rate of evolution and strain turnover in the infant gut compared with healthy adults, with the mother-infant transition at delivery being a particularly dynamic period in which gene loss dominates. Within a few months after birth, these dynamics stabilize, and gene gains become increasingly frequent as the microbiome matures. We furthermore find that evolutionary changes in infants show signatures of being seeded by a mixture of de novo mutations and transmissions of pre-evolved lineages from the broader family. Several of these evolutionary changes occur in parallel across infants, highlighting candidate genes that may play important roles in the development of the infant gut microbiome. Our results point to a picture of a volatile infant gut microbiome characterized by rapid evolutionary and ecological change in the early days of life.

Evaluating supervised and unsupervised background noise correction in human gut microbiome data.

The ability to predict human phenotypes and identify biomarkers of disease from metagenomic data is crucial for the development of therapeutics for microbiome-associated diseases. However, metagenomic data is commonly affected by technical variables unrelated to the phenotype of interest, such as sequencing protocol, which can make it difficult to predict phenotype and find biomarkers of disease. Supervised methods to correct for background noise, originally designed for gene expression and RNA-seq data, are commonly applied to microbiome data but may be limited because they cannot account for unmeasured sources of variation. Unsupervised approaches address this issue, but current methods are limited because they are ill-equipped to deal with the unique aspects of microbiome data, which is compositional, highly skewed, and sparse. We perform a comparative analysis of the ability of different denoising transformations in combination with supervised correction methods as well as an unsupervised principal component correction approach that is presently used in other domains but has not been applied to microbiome data to date. We find that the unsupervised principal component correction approach has comparable ability in reducing false discovery of biomarkers as the supervised approaches, with the added benefit of not needing to know the sources of variation apriori. However, in prediction tasks, it appears to only improve prediction when technical variables contribute to the majority of variance in the data. As new and larger metagenomic datasets become increasingly available, background noise correction will become essential for generating reproducible microbiome analyses.

The effect of gastric acid suppression on probiotic colonization in a double blinded randomized clinical trial.

BACKGROUND & AIMS: Probiotics contain living microorganisms consumed for their putative benefits on the intestinal microbiota and general health and a concept is emerging to use probiotic as a therapeutic intervention to reduce proton pump inhibitors (PPIs) negative effects, but data is lacking. The use of PPIs can result in disordered gut microbiota, leading to a risk of enteric infections. PPIs are frequently prescribed in the general practice setting for gastroesophageal reflux disease (GERD), peptic ulcer disease, and related conditions. Despite the availability and widespread use of probiotics and acid-suppressing medications, the effect of PPIs-induced gastric acid suppression on the survival and colonization of probiotics bacterial species is currently unclear. We hypothesized that gastric acid suppression may improve intestinal colonization of probiotics bacterial species and probiotic intervention may have a potential role in mitigating untoward effects of PPI. METHODS: In a randomized, double-blind, placebo-controlled study, healthy subjects were given either proton pump inhibitor (PPI, n = 15) or placebo (n = 15) over 6 weeks. All subjects then consumed multi-strain probiotics from weeks 2-6. Thirty participants (10 males, 20 females, age range: 18-56 years) were enrolled in the study. Shotgun metagenomic sequencing and untargeted metabolomics analyses were performed on stool samples collected at week 0, 2, and 6. RESULTS: Short term PPI treatment increased the microbial abundance of Streptococcaceae (p = 0.004), Leuconostacaceae (p = 0.001), and Pasteurellaceae (p = 0.020) at family level and corresponding genus levels. The metabolomic analysis of the stools revealed a change in 10 metabolites where Gly Arg Val and phenylacetic acid were consistently increased compared to the baseline. Probiotic intervention inhibited PPI-induced microbial changes such as a decrease in Leuconostacaceae family (p = 0.01) and led to an increase in metabolite 1H-Indole-4-carbaldehyde. Notably, PPI enhanced the colonization of certain probiotic bacterial species like Streptococcus thermophilus (p < 0.05) along with other species present in the multi-strain probiotic. CONCLUSION: Acid suppression enhanced certain probiotic associated bacterial colonization and probiotics in turn suppressed PPI-mediated intestinal microbial alterations. Thus, probiotics in combination with PPI might be a beneficial strategy that allows probiotic colonization and suppress PPI-induced microbial perturbations. CLINICAL TRIALS. GOV, NUMBER: NCT03327051.

Comparative Population Genetics in the Human Gut Microbiome.

Genetic variation in the human gut microbiome is responsible for conferring a number of crucial phenotypes like the ability to digest food and metabolize drugs. Yet, our understanding of how this variation arises and is maintained remains relatively poor. Thus, the microbiome remains a largely untapped resource, as the large number of coexisting species in the microbiome presents a unique opportunity to compare and contrast evolutionary processes across species to identify universal trends and deviations. Here we outline features of the human gut microbiome that, while not unique in isolation, as an assemblage make it a system with unparalleled potential for comparative population genomics studies. We consciously take a broad view of comparative population genetics, emphasizing how sampling a large number of species allows researchers to identify universal evolutionary dynamics in addition to new genes, which can then be leveraged to identify exceptional species that deviate from general patterns. To highlight the potential power of comparative population genetics in the microbiome, we reanalyze patterns of purifying selection across ∼40 prevalent species in the human gut microbiome to identify intriguing trends which highlight functional categories in the microbiome that may be under more or less constraint.

Longitudinal linked-read sequencing reveals ecological and evolutionary responses of a human gut microbiome during antibiotic treatment.

Gut microbial communities can respond to antibiotic perturbations by rapidly altering their taxonomic and functional composition. However, little is known about the strain-level processes that drive this collective response. Here, we characterize the gut microbiome of a single individual at high temporal and genetic resolution through a period of health, disease, antibiotic treatment, and recovery. We used deep, linked-read metagenomic sequencing to track the longitudinal trajectories of thousands of single nucleotide variants within 36 species, which allowed us to contrast these genetic dynamics with the ecological fluctuations at the species level. We found that antibiotics can drive rapid shifts in the genetic composition of individual species, often involving incomplete genome-wide sweeps of pre-existing variants. These genetic changes were frequently observed in species without obvious changes in species abundance, emphasizing the importance of monitoring diversity below the species level. We also found that many sweeping variants quickly reverted to their baseline levels once antibiotic treatment had concluded, demonstrating that the ecological resilience of the microbiota can sometimes extend all the way down to the genetic level. Our results provide new insights into the population genetic forces that shape individual microbiomes on therapeutically relevant timescales, with potential implications for personalized health and disease.

Detection of hard and soft selective sweeps from Drosophila melanogaster population genomic data.

Whether hard sweeps or soft sweeps dominate adaptation has been a matter of much debate. Recently, we developed haplotype homozygosity statistics that (i) can detect both hard and soft sweeps with similar power and (ii) can classify the detected sweeps as hard or soft. The application of our method to population genomic data from a natural population of Drosophila melanogaster (DGRP) allowed us to rediscover three known cases of adaptation at the loci Ace, Cyp6g1, and CHKov1 known to be driven by soft sweeps, and detected additional candidate loci for recent and strong sweeps. Surprisingly, all of the top 50 candidates showed patterns much more consistent with soft rather than hard sweeps. Recently, Harris et al. 2018 criticized this work, suggesting that all the candidate loci detected by our haplotype statistics, including the positive controls, are unlikely to be sweeps at all and that instead these haplotype patterns can be more easily explained by complex neutral demographic models. They also claim that these neutral non-sweeps are likely to be hard instead of soft sweeps. Here, we reanalyze the DGRP data using a range of complex admixture demographic models and reconfirm our original published results suggesting that the majority of recent and strong sweeps in D. melanogaster are first likely to be true sweeps, and second, that they do appear to be soft. Furthermore, we discuss ways to take this work forward given that most demographic models employed in such analyses are necessarily too simple to capture the full demographic complexity, while more realistic models are unlikely to be inferred correctly because they require a large number of free parameters.

Population Genetics in the Human Microbiome.

While the human microbiome's structure and function have been extensively studied, its within-species genetic diversity is less well understood. However, genetic mutations in the microbiome can confer biomedically relevant traits, such as the ability to extract nutrients from food, metabolize drugs, evade antibiotics, and communicate with the host immune system. The population genetic processes by which these traits evolve are complex, in part due to interacting ecological and evolutionary forces in the microbiome. Advances in metagenomic sequencing, coupled with bioinformatics tools and population genetic models, facilitate quantification of microbiome genetic variation and inferences about how this diversity arises, evolves, and correlates with traits of both microbes and hosts. In this review, we explore the population genetic forces (mutation, recombination, drift, and selection) that shape microbiome genetic diversity within and between hosts, as well as efforts towards predictive models that leverage microbiome genetics.

Evolutionary dynamics of bacteria in the gut microbiome within and across hosts.

Gut microbiota are shaped by a combination of ecological and evolutionary forces. While the ecological dynamics have been extensively studied, much less is known about how species of gut bacteria evolve over time. Here, we introduce a model-based framework for quantifying evolutionary dynamics within and across hosts using a panel of metagenomic samples. We use this approach to study evolution in approximately 40 prevalent species in the human gut. Although the patterns of between-host diversity are consistent with quasi-sexual evolution and purifying selection on long timescales, we identify new genealogical signatures that challenge standard population genetic models of these processes. Within hosts, we find that genetic differences that accumulate over 6-month timescales are only rarely attributable to replacement by distantly related strains. Instead, the resident strains more commonly acquire a smaller number of putative evolutionary changes, in which nucleotide variants or gene gains or losses rapidly sweep to high frequency. By comparing these mutations with the typical between-host differences, we find evidence that some sweeps may be seeded by recombination, in addition to new mutations. However, comparisons of adult twins suggest that replacement eventually overwhelms evolution over multi-decade timescales, hinting at fundamental limits to the extent of local adaptation. Together, our results suggest that gut bacteria can evolve on human-relevant timescales, and they highlight the connections between these short-term evolutionary dynamics and longer-term evolution across hosts.

Detection and Classification of Hard and Soft Sweeps from Unphased Genotypes by Multilocus Genotype Identity.

Positive natural selection can lead to a decrease in genomic diversity at the selected site and at linked sites, producing a characteristic signature of elevated expected haplotype homozygosity. These selective sweeps can be hard or soft. In the case of a hard selective sweep, a single adaptive haplotype rises to high population frequency, whereas multiple adaptive haplotypes sweep through the population simultaneously in a soft sweep, producing distinct patterns of genetic variation in the vicinity of the selected site. Measures of expected haplotype homozygosity have previously been used to detect sweeps in multiple study systems. However, these methods are formulated for phased haplotype data, typically unavailable for nonmodel organisms, and some may have reduced power to detect soft sweeps due to their increased genetic diversity relative to hard sweeps. To address these limitations, we applied the H12 and H2/H1 statistics proposed in 2015 by Garud et al., which have power to detect both hard and soft sweeps, to unphased multilocus genotypes, denoting them as G12 and G2/G1. G12 (and the more direct expected homozygosity analog to H12, denoted G123) has comparable power to H12 for detecting both hard and soft sweeps. G2/G1 can be used to classify hard and soft sweeps analogously to H2/H1, conditional on a genomic region having high G12 or G123 values. The reason for this power is that, under random mating, the most frequent haplotypes will yield the most frequent multilocus genotypes. Simulations based on parameters compatible with our recent understanding of human demographic history suggest that expected homozygosity methods are best suited for detecting recent sweeps, and increase in power under recent population expansions. Finally, we find candidates for selective sweeps within the 1000 Genomes CEU, YRI, GIH, and CHB populations, which corroborate and complement existing studies.

Structural Variants and Selective Sweep Foci Contribute to Insecticide Resistance in the Drosophila Genetic Reference Panel.

Patterns of nucleotide polymorphism within populations of Drosophila melanogaster suggest that insecticides have been the selective agents driving the strongest recent bouts of positive selection. However, there is a need to explicitly link selective sweeps to the particular insecticide phenotypes that could plausibly account for the drastic selective responses that are observed in these non-target insects. Here, we screen the Drosophila Genetic Reference Panel with two common insecticides; malathion (an organophosphate) and permethrin (a pyrethroid). Genome-wide association studies map survival on malathion to two of the largest sweeps in the D. melanogaster genome; Ace and Cyp6g1 Malathion survivorship also correlates with lines which have high levels of Cyp12d1, Jheh1 and Jheh2 transcript abundance. Permethrin phenotypes map to the largest cluster of P450 genes in the Drosophila genome, however in contrast to a selective sweep driven by insecticide use, the derived allele seems to be associated with susceptibility. These results underscore previous findings that highlight the importance of structural variation to insecticide phenotypes: Cyp6g1 exhibits copy number variation and transposable element insertions, Cyp12d1 is tandemly duplicated, the Jheh loci are associated with a Bari1 transposable element insertion, and a Cyp6a17 deletion is associated with susceptibility.

Elevated Linkage Disequilibrium and Signatures of Soft Sweeps Are Common in Drosophila melanogaster.

The extent to which selection and demography impact patterns of genetic diversity in natural populations of Drosophila melanogaster is yet to be fully understood. We previously observed that linkage disequilibrium (LD) at scales of ∼10 kb in the Drosophila Genetic Reference Panel (DGRP), consisting of 145 inbred strains from Raleigh, North Carolina, measured both between pairs of sites and as haplotype homozygosity, is elevated above neutral demographic expectations. We also demonstrated that signatures of strong and recent soft sweeps are abundant. However, the extent to which these patterns are specific to this derived and admixed population is unknown. It is also unclear whether these patterns are a consequence of the extensive inbreeding performed to generate the DGRP data. Here we analyze LD statistics in a sample of >100 fully-sequenced strains from Zambia; an ancestral population to the Raleigh population that has experienced little to no admixture and was generated by sequencing haploid embryos rather than inbred strains. We find an elevation in long-range LD and haplotype homozygosity compared to neutral expectations in the Zambian sample, thus showing the elevation in LD is not specific to the DGRP data set. This elevation in LD and haplotype structure remains even after controlling for possible confounders including genomic inversions, admixture, population substructure, close relatedness of individual strains, and recombination rate variation. Furthermore, signatures of partial soft sweeps similar to those found in the DGRP as well as partial hard sweeps are common in Zambia. These results suggest that while the selective forces and sources of adaptive mutations may differ in Zambia and Raleigh, elevated long-range LD and signatures of soft sweeps are generic in D. melanogaster.

The population genetics of drug resistance evolution in natural populations of viral, bacterial and eukaryotic pathogens.

Drug resistance is a costly consequence of pathogen evolution and a major concern in public health. In this review, we show how population genetics can be used to study the evolution of drug resistance and also how drug resistance evolution is informative as an evolutionary model system. We highlight five examples from diverse organisms with particular focus on: (i) identifying drug resistance loci in the malaria parasite Plasmodium falciparum using the genomic signatures of selective sweeps, (ii) determining the role of epistasis in drug resistance evolution in influenza, (iii) quantifying the role of standing genetic variation in the evolution of drug resistance in HIV, (iv) using drug resistance mutations to study clonal interference dynamics in tuberculosis and (v) analysing the population structure of the core and accessory genome of Staphylococcus aureus to understand the spread of methicillin resistance. Throughout this review, we discuss the uses of sequence data and population genetic theory in studying the evolution of drug resistance.

Enhancing the mathematical properties of new haplotype homozygosity statistics for the detection of selective sweeps.

Soft selective sweeps represent an important form of adaptation in which multiple haplotypes bearing adaptive alleles rise to high frequency. Most statistical methods for detecting selective sweeps from genetic polymorphism data, however, have focused on identifying hard selective sweeps in which a favored allele appears on a single haplotypic background; these methods might be underpowered to detect soft sweeps. Among exceptions is the set of haplotype homozygosity statistics introduced for the detection of soft sweeps by Garud et al. (2015). These statistics, examining frequencies of multiple haplotypes in relation to each other, include H12, a statistic designed to identify both hard and soft selective sweeps, and H2/H1, a statistic that conditional on high H12 values seeks to distinguish between hard and soft sweeps. A challenge in the use of H2/H1 is that its range depends on the associated value of H12, so that equal H2/H1 values might provide different levels of support for a soft sweep model at different values of H12. Here, we enhance the H12 and H2/H1 haplotype homozygosity statistics for selective sweep detection by deriving the upper bound on H2/H1 as a function of H12, thereby generating a statistic that normalizes H2/H1 to lie between 0 and 1. Through a reanalysis of resequencing data from inbred lines of Drosophila, we show that the enhanced statistic both strengthens interpretations obtained with the unnormalized statistic and leads to empirical insights that are less readily apparent without the normalization.