Duy Tin Truong

MetaPhlAn2 for enhanced metagenomic taxonomic profiling.

 Profiling of all domains of life. Marker and quasi-marker genes are now identified not only for microbes (Bacteria and Archaea), but also for viruses and Eukaryotic microbes (Fungi, Protozoa) that are crucial components of microbial communities.  A 6-fold increase in the number of considered species. Markers are now identified from >16,000 reference genomes and >7,000 unique species, dramatically expanding the comprehensiveness of the method. The new pipeline for identifying marker genes is also scalable to the quickly increasing number of reference genomes. See Supplementary Tables 1-3.  Introduction of the concept of quasi-markers, allowing more comprehensive and accurate profiling. For species with less than 200 markers, MetaPhlAn2 adopts additional quasi-marker sequences (Supplementary Note 2) that are occasionally present in other genomes (because of vertical conservation or horizontal transfer). At profiling time, if no other markers of the potentially confounding species are detected, the corresponding quasi-local markers are used to improve the quality and accuracy of the profiling.  Addition of strain-specific barcoding for microbial strain tracking. MetaPhlAn2 includes a completely new feature that exploits marker combinations to perform species-specific and genus-specific “barcoding” for strains in metagenomic samples (Supplementary Note 7). This feature can be used for culture-free pathogen tracking in epidemiology studies and strain tracking across microbiome samples. See Supplementary Figs. 12-20.  Strain-level identification for organisms with sequenced genomes. For the case in which a microbiome includes strains that are very close to one of those already sequenced, MetaPhlAn2 is now able to identify such strains and readily reports their abundances. See Supplementary Note 7, Supplementary Table 13, and Supplementary Fig. 21.  Improvement of false positive and false negative rates. Improvements in the underlying pipeline for identifying marker genes (including the increment of the adopted genomes and the use of quasi-markers) and the profiling procedure resulted in much improved quantitative performances (higher correlation with true abundances, lower false positive and false negative rates). See the validation on synthetic metagenomes in Supplementary Note 4.  Estimation of the percentage of reads mapped against known reference genomes. MetaPhlAn2 is now able to estimate the number of reads that would map against genomes of each clade detected as present and for which an estimation of its relative abundance is provided by the default output. See Supplementary Note 3 for details.  Integration of MetaPhlAn with post-processing and visualization tools. The MetaPhlAn2 package now includes a set of post-processing and visualization tools (“utils” subfolder of the MetaPhlAn2 repository). Multiple MetaPhlAn profiles can in fact be merged in an abundance table (“merge_metaphlan_tables.py”), exported as BIOM files, visualized as heatmap (“metaphlan_hclust_heatmap.py” or the integrated “hclust2” package), GraPhlAn plots (“export2graphlan.py” and the GraPhlAn package1), Krona2 plots (“metaphlan2krona.py”), and single microbe barplot across samples and conditions (“plot_bug.py”).

Strain-level microbial epidemiology and population genomics from shotgun metagenomics

Identifying microbial strains and characterizing their functional potential is essential for pathogen discovery, epidemiology and population genomics. We present pangenome-based phylogenomic analysis (PanPhlAn; http://segatalab.cibio.unitn.it/tools/panphlan), a tool that uses metagenomic data to achieve strain-level microbial profiling resolution. PanPhlAn recognized outbreak strains, produced the largest strain-level population genomic study of human-associated bacteria and, in combination with metatranscriptomics, profiled the transcriptional activity of strains in complex communities.

Microbial strain-level population structure and genetic diversity from metagenomes

Among the human health conditions linked to microbial communities, phenotypes are often associated with only a subset of strains within causal microbial groups. Although it has been critical for decades in microbial physiology to characterize individual strains, this has been challenging when using culture-independent high-throughput metagenomics. We introduce StrainPhlAn, a novel metagenomic strain identification approach, and apply it to characterize the genetic structure of thousands of strains from more than 125 species in more than 1500 gut metagenomes drawn from populations spanning North and South American, European, Asian, and African countries. The method relies on per-sample dominant sequence variant reconstruction within species-specific marker genes. It identified primarily subject-specific strain variants (<5% inter-subject strain sharing), and we determined that a single strain typically dominated each species and was retained over time (for >70% of species). Microbial population structure was correlated in several distinct ways with the geographic structure of the host population. In some cases, discrete subspecies (e.g., for Eubacterium rectale and Prevotella copri) or continuous microbial genetic variations (e.g., for Faecalibacterium prausnitzii) were associated with geographically distinct human populations, whereas few strains occurred in multiple unrelated cohorts. We further estimated the genetic variability of gut microbes, with Bacteroides species appearing remarkably consistent (0.45% median number of nucleotide variants between strains), whereas P. copri was among the most plastic gut colonizers. We thus characterize here the population genetics of previously inaccessible intestinal microbes, providing a comprehensive strain-level genetic overview of the gut microbial diversity.

Machine Learning Meta-analysis of Large Metagenomic Datasets: Tools and Biological Insights.

Shotgun metagenomic analysis of the human associated microbiome provides a rich set of microbial features for prediction and biomarker discovery in the context of human diseases and health conditions. However, the use of such high-resolution microbial features presents new challenges, and validated computational tools for learning tasks are lacking. Moreover, classification rules have scarcely been validated in independent studies, posing questions about the generality and generalization of disease-predictive models across cohorts. In this paper, we comprehensively assess approaches to metagenomics-based prediction tasks and for quantitative assessment of the strength of potential microbiome-phenotype associations. We develop a computational framework for prediction tasks using quantitative microbiome profiles, including species-level relative abundances and presence of strain-specific markers. A comprehensive meta-analysis, with particular emphasis on generalization across cohorts, was performed in a collection of 2424 publicly available metagenomic samples from eight large-scale studies. Cross-validation revealed good disease-prediction capabilities, which were in general improved by feature selection and use of strain-specific markers instead of species-level taxonomic abundance. In cross-study analysis, models transferred between studies were in some cases less accurate than models tested by within-study cross-validation. Interestingly, the addition of healthy (control) samples from other studies to training sets improved disease prediction capabilities. Some microbial species (most notably Streptococcus anginosus) seem to characterize general dysbiotic states of the microbiome rather than connections with a specific disease. Our results in modelling features of the “healthy” microbiome can be considered a first step toward defining general microbial dysbiosis. The software framework, microbiome profiles, and metadata for thousands of samples are publicly available at http://segatalab.cibio.unitn.it/tools/metaml.

Studying Vertical Microbiome Transmission from Mothers to Infants by Strain-Level Metagenomic Profiling.

Early infant exposure is important in the acquisition and ultimate development of a healthy infant microbiome. There is increasing support for the idea that the maternal microbial reservoir is a key route of microbial transmission, and yet much is inferred from the observation of shared species in mother and infant. The presence of common species, per se, does not necessarily equate to vertical transmission, as species exhibit considerable strain heterogeneity. It is therefore imperative to assess whether shared microbes belong to the same genetic variant (i.e., strain) to support the hypothesis of vertical transmission. Here we demonstrate the potential of shotgun metagenomics and strain-level profiling to identify vertical transmission events. Combining these data with metatranscriptomics, we show that it is possible not only to identify and track the fate of microbes in the early infant microbiome but also to investigate the actively transcribing members of the community. These approaches will ultimately provide important insights into the acquisition, development, and community dynamics of the infant microbiome. ABSTRACT The gut microbiome becomes shaped in the first days of life and continues to increase its diversity during the first months. Links between the configuration of the infant gut microbiome and infant health are being shown, but a comprehensive strain-level assessment of microbes vertically transmitted from mother to infant is still missing. We collected fecal and breast milk samples from multiple mother-infant pairs during the first year of life and applied shotgun metagenomic sequencing followed by computational strain-level profiling. We observed that several specific strains, including those of Bifidobacterium bifidum, Coprococcus comes, and Ruminococcus bromii, were present in samples from the same mother-infant pair, while being clearly distinct from those carried by other pairs, which is indicative of vertical transmission. We further applied metatranscriptomics to study the in vivo gene expression of vertically transmitted microbes and found that transmitted strains of Bacteroides and Bifidobacterium species were transcriptionally active in the guts of both adult and infant. By combining longitudinal microbiome sampling and newly developed computational tools for strain-level microbiome analysis, we demonstrated that it is possible to track the vertical transmission of microbial strains from mother to infants and to characterize their transcriptional activity. Our work provides the foundation for larger-scale surveys to identify the routes of vertical microbial transmission and its influence on postinfancy microbiome development. IMPORTANCE Early infant exposure is important in the acquisition and ultimate development of a healthy infant microbiome. There is increasing support for the idea that the maternal microbial reservoir is a key route of microbial transmission, and yet much is inferred from the observation of shared species in mother and infant. The presence of common species, per se, does not necessarily equate to vertical transmission, as species exhibit considerable strain heterogeneity. It is therefore imperative to assess whether shared microbes belong to the same genetic variant (i.e., strain) to support the hypothesis of vertical transmission. Here we demonstrate the potential of shotgun metagenomics and strain-level profiling to identify vertical transmission events. Combining these data with metatranscriptomics, we show that it is possible not only to identify and track the fate of microbes in the early infant microbiome but also to investigate the actively transcribing members of the community. These approaches will ultimately provide important insights into the acquisition, development, and community dynamics of the infant microbiome.

The reproductive tracts of two malaria vectors are populated by a core microbiome and by gender- and swarm-enriched microbial biomarkers

Microbes play key roles in shaping the physiology of insects and can influence behavior, reproduction and susceptibility to pathogens. In Sub-Saharan Africa, two major malaria vectors, Anopheles gambiae and An. coluzzii, breed in distinct larval habitats characterized by different microorganisms that might affect their adult physiology and possibly Plasmodium transmission. We analyzed the reproductive microbiomes of male and female An. gambiae and An. coluzzii couples collected from natural mating swarms in Burkina Faso. 16S rRNA sequencing on dissected tissues revealed that the reproductive tracts harbor a complex microbiome characterized by a large core group of bacteria shared by both species and all reproductive tissues. Interestingly, we detected a significant enrichment of several gender-associated microbial biomarkers in specific tissues, and surprisingly, similar classes of bacteria in males captured from one mating swarm, suggesting that these males originated from the same larval breeding site. Finally, we identified several endosymbiotic bacteria, including Spiroplasma, which have the ability to manipulate insect reproductive success. Our study provides a comprehensive analysis of the reproductive microbiome of important human disease vectors, and identifies a panel of core and endosymbiotic bacteria that can be potentially exploited to interfere with the transmission of malaria parasites by the Anopheles mosquito.

Unexplored diversity and strain-level structure of the skin microbiome associated with psoriasis

Psoriasis is an immune-mediated inflammatory skin disease that has been associated with cutaneous microbial dysbiosis by culture-dependent investigations and rRNA community profiling. We applied, for the first time, high-resolution shotgun metagenomics to characterise the microbiome of psoriatic and unaffected skin from 28 individuals. We demonstrate psoriatic ear sites have a decreased diversity and psoriasis is associated with an increase in Staphylococcus, but overall the microbiomes of psoriatic and unaffected sites display few discriminative features at the species level. Finer strain-level analysis reveals strain heterogeneity colonisation and functional variability providing the intriguing hypothesis of psoriatic niche-specific strain adaptation or selection. Furthermore, we accessed the poorly characterised, but abundant, clades with limited sequence information in public databases, including uncharacterised Malassezia spp. These results highlight the skins hidden diversity and suggests strain-level variations could be key determinants of the psoriatic microbiome. This illustrates the need for high-resolution analyses, particularly when identifying therapeutic targets. This work provides a baseline for microbiome studies in relation to the pathogenesis of psoriasis.Psoriasis: investigating microbial diversityAnalysing microbial populations on the skin provides an insight into the diversity of microbes associated with psoriasis. Nicola Segata and colleagues at the University of Trento, Italy, used genetic analysis to compare the microbial populations on regions of skin affected and unaffected by psoriasis. Staphylococcus bacteria were more prevalent in psoriasis, but there was little clearly defined difference in microbial species on psoriasis-affected and unaffected skin. There was, however, decreased microbial diversity on psoriatic ear sites. Deeper strain-level computational analysis suggested that psoriasis could offer niche locations for colonisation by specific strains of staphylococci and propionibacteria. The results highlight the diversity of microbial populations on the skin, and the need for larger cohorts to build on the baseline data now established. Further studies might help identify targets for treating skin bacteria associated with psoriasis.

Uncovering oral Neisseria tropism and persistence using metagenomic sequencing

Microbial epidemiology and population genomics have previously been carried out near-exclusively for organisms grown in vitro. Metagenomics helps to overcome this limitation, but it is still challenging to achieve strain-level characterization of microorganisms from culture-independent data with sufficient resolution for epidemiological modelling. Here, we have developed multiple complementary approaches that can be combined to profile and track individual microbial strains. To specifically profile highly recombinant neisseriae from oral metagenomes, we integrated four metagenomic analysis techniques: single nucleotide polymorphisms in the clades core genome, DNA uptake sequence signatures, metagenomic multilocus sequence typing and strain-specific marker genes. We applied these tools to 520 oral metagenomes from the Human Microbiome Project, finding evidence of site tropism and temporal intra-subject strain retention. Although the opportunistic pathogen Neisseria meningitidis is enriched for colonization in the throat, N. flavescens and N. subflava populate the tongue dorsum, and N. sicca, N. mucosa and N. elongata the gingival plaque. The buccal mucosa appeared as an intermediate ecological niche between the plaque and the tongue. The resulting approaches to metagenomic strain profiling are generalizable and can be extended to other organisms and microbiomes across environments.

Accessible, curated metagenomic data through ExperimentHub

Affiliations: 1 Centre for Integrative Biology, University of Trento, Trento, Italy 2 Institute for Implementation Science and Population Health, City University of New York School of Public Health, New York, New York, United States of America 3 Roswell Park Cancer Institute, University of Buffalo, Buffalo, New York, United States of America 4 Department of Global Health and Social Medicine, King’s College London 5 Biostatistics Department, Harvard School of Public Health, Boston, Massachusetts, United States of America 6 The Broad Institute, Cambridge, Massachusetts, United States of America

Mother-to-Infant Microbial Transmission from Different Body Sites Shapes the Developing Infant Gut Microbiome

Summary The acquisition and development of the infant microbiome are key to establishing a healthy host-microbiome symbiosis. The maternal microbial reservoir is thought to play a crucial role in this process. However, the source and transmission routes of the infant pioneering microbes are poorly understood. To address this, we longitudinally sampled the microbiome of 25 mother-infant pairs across multiple body sites from birth up to 4 months postpartum. Strain-level metagenomic profiling showed a rapid influx of microbes at birth followed by strong selection during the first few days of life. Maternal skin and vaginal strains colonize only transiently, and the infant continues to acquire microbes from distinct maternal sources after birth. Maternal gut strains proved more persistent in the infant gut and ecologically better adapted than those acquired from other sources. Together, these data describe the mother-to-infant microbiome transmission routes that are integral in the development of the infant microbiome.