Metagenomics: a path to understanding the gut microbiome

The gut microbiome is a major determinant of host health, yet it is only in the last 2 decades that the advent of next-generation sequencing has enabled it to be studied at a genomic level. Shotgun sequencing is beginning to provide insight into the prokaryotic as well as eukaryotic and viral components of the gut community, revealing not just their taxonomy, but also the functions encoded by their collective metagenome. This revolution in understanding is being driven by continued development of sequencing technologies and in consequence necessitates reciprocal development of computational approaches that can adapt to the evolving nature of sequence datasets. In this review, we provide an overview of current bioinformatic strategies for handling metagenomic sequence data and discuss their strengths and limitations. We then go on to discuss key technological developments that have the potential to once again revolutionise the way we are able to view and hence understand the microbiome.

Microbe Finder (MiFi®): Implementation of an Interactive Pathogen Detection Tool in Metagenomic Sequence Data

Agricultural high throughput diagnostics need to be fast, accurate and have multiplexing capacity. Metagenomic sequencing is being widely evaluated for plant and animal diagnostics. Bioinformatic analysis of metagenomic sequence data has been a bottleneck for diagnostic analysis due to the size of the data files. Most available tools for analyzing high-throughput sequencing (HTS) data require that the user have computer coding skills and access to high-performance computing. To overcome constraints to most sequencing-based diagnostic pipelines today, we have developed Microbe Finder (MiFi®). MiFi® is a web application for quick detection and identification of known pathogen species/strains in raw, unassembled HTS metagenomic data. HTS-based diagnostic tools developed through MiFi® must pass rigorous validation, which is outlined in this manuscript. MiFi® allows researchers to collaborate in the development and validation of HTS-based diagnostic assays using MiProbe™, a platform used for developing pathogen-specific e-probes. Validated e-probes are made available to diagnosticians through MiDetect™. Here we describe the e-probe development, curation and validation process of MiFi® using grapevine pathogens as a model system. MiFi® can be used with any pathosystem and HTS platform after e-probes have been validated.

A Need for Improved Cellulase Identification from Metagenomic Sequence Data

ABSTRACT Improved sequencing technologies and the maturation of metagenomic approaches allow the identification of gene variants with potential industrial applications, including cellulases. Cellulase identification from metagenomic environmental surveys is complicated by inconsistent nomenclature and multiple categorization systems. Here, we summarize the current classification and nomenclature systems, with recommendations for improvements to these systems. Addressing the issues described will strengthen the annotation of cellulose-active enzymes from environmental sequence data sets—a rapidly growing resource in environmental and applied microbiology.

Novel Approach for Microbiome Analysis Using Bacterial Replication Rates and Causal Inference with Applications

Motivation: Metagenomics sequencing data can be used to compute not just the relative abundance profile, but also the replication rates of every taxon in the microbiome sample. We investigate how the dynamics implied by the replication rates can be used to understand the antibiotic response in microbiomes, given the significant variation in the types of antibiotics and the types of response by different taxa. The analysis is further expanded by factoring in the resistome of the microbiomes, which can be readily profiled from the metagenomic sequence data. The fact that some antibiotics such as β -lactams target replicating cells makes it even more critical to use replication rates to analyze the antibiotic response. Results: We introduce a novel approach for metagenomic analysis that integrates microbial community profiling, replication rate calculation, and causal structural learning to analyze the antibiotic response. First, we developed PeTRi, which involves efficient cluster computation of bacterial replication rates from metagenomic sequence data. Second, we integrate the abundance profile, replication profile, resistome profile, and environmental variables to perform causality analysis. Finally, we applied the integrated analysis to the data from an infant gut microbiome study. Conclusions from our analysis are as follows: (i) Microbes tend to lower their replication rates in response to β -lactams; (ii) The presence of antibiotic resistance genes combined with the causality analysis strongly suggest that genes fosA5, oqxA, kpnF, arnA, and acrA provides resistance for the taxon K. pneumoniae, allowing it to replicate and dominate the microbiome after the drug ticarcillin-clavulanate was administered; and (iii) Human and donor milk strongly influence the resistome of the infant gut microbiome.

Time Series Resolution of the Fish Necrobiome Reveals a Decomposer Succession Involving Toxigenic Bacterial Pathogens

ABSTRACT Despite progress understanding microbial communities involved in terrestrial vertebrate decomposition, little is known about the microbial decomposition of aquatic vertebrates from a functional and environmental context. Here, we analyzed temporal changes in the “necrobiome” of rainbow darters, which are common North American fish that are sensitive indicators of water quality. By combining 16S rRNA gene and shotgun metagenomic sequence data from four time points, we studied the progression of decomposers from both taxonomic and functional perspectives. The 16S rRNA gene profiles revealed strong community succession, with early decomposition stages associated with Aeromonas and Clostridium taxa and later stages dominated by members of the Rikenellaceae (i.e., Alistipes/Acetobacteroides genera). These results were reproducible and independent of environmental perturbation, given that exposure to wastewater treatment plant effluent did not substantially influence the necrobiome composition of fish or the associated water sample microbiota. Metagenomic analysis revealed significant changes throughout decomposition in degradation pathways for amino acids, carbohydrates/glycans, and other compounds, in addition to putrefaction pathways for production of putrescine, cadaverine, and indole. Binning of contigs confirmed a predominance of Aeromonas genome assemblies, including those from novel strains related to the pathogen Aeromonas veronii. These bins of Aeromonas genes also encoded known hemolysin toxins (e.g., aerolysin) that were particularly abundant early in the process, potentially contributing to host cell lysis during decomposition. Overall, our results demonstrate that wild-caught fish have a reproducible decomposer succession and that the fish necrobiome serves as a potential source of putative pathogens and toxigenic bacteria. IMPORTANCE The microbial decomposition of animal tissues is an important ecological process that impacts nutrient cycling in natural environments. We studied the microbial decomposition of a common North American fish (rainbow darters) over four time points, combining 16S rRNA gene and shotgun metagenomic sequence data to obtain both taxonomic and functional perspectives. Our data revealed a strong community succession that was reproduced across different fish and environments. Decomposition time point was the main driver of community composition and functional potential; fish environmental origin (upstream or downstream of a wastewater treatment plant) had a secondary effect. We also identified strains related to the putative pathogen Aeromonas veronii as dominant members of the decomposition community. These bacteria peaked early in decomposition and coincided with the metagenomic abundance of hemolytic toxin genes. Our work reveals a strong decomposer succession in wild-caught fish, providing functional and taxonomic insights into the vertebrate necrobiome.

Improved metagenomic analysis with Kraken 2

Although Kraken’s k-mer-based approach provides a fast taxonomic classification of metagenomic sequence data, its large memory requirements can be limiting for some applications. Kraken 2 improves upon Kraken 1 by reducing memory usage by 85%, allowing greater amounts of reference genomic data to be used, while maintaining high accuracy and increasing speed fivefold. Kraken 2 also introduces a translated search mode, providing increased sensitivity in viral metagenomics analysis.

The composition and functional protein subsystems of the human nasal microbiome in granulomatosis with polyangiitis: a pilot study

Background Ear, nose and throat involvement in granulomatosis with polyangiitis (GPA) is frequently the initial disease manifestation. Previous investigations have observed a higher prevalence of Staphylococcus aureus in patients with GPA, and chronic nasal carriage has been linked with an increased risk of disease relapse. In this cross-sectional study, we investigated changes in the nasal microbiota including a detailed analysis of Staphylococcus spp. by shotgun metagenomics in patients with active and inactive granulomatosis with polyangiitis (GPA). Shotgun metagenomic sequence data were also used to identify protein-encoding genes within the SEED database, and the abundance of proteins then correlated with the presence of bacterial species on an annotated heatmap. Results The presence of S. aureus in the nose as assessed by culture was more frequently detected in patients with active GPA (66.7%) compared with inactive GPA (34.1%). Beta diversity analysis of nasal microbiota by bacterial 16S rRNA profiling revealed a different composition between GPA patients and healthy controls (P = 0.039). Beta diversity analysis of shotgun metagenomic sequence data for Staphylococcus spp. revealed a different composition between active GPA patients and healthy controls and disease controls (P = 0.0007 and P = 0.0023, respectively), and between healthy controls and inactive GPA patients and household controls (P = 0.0168 and P = 0.0168, respectively). Patients with active GPA had a higher abundance of S. aureus, mirroring the culture data, while healthy controls had a higher abundance of S. epidermidis. Staphylococcus pseudintermedius, generally assumed to be a pathogen of cats and dogs, showed an abundance of 13% among the Staphylococcus spp. in our cohort. During long-term follow-up of patients with inactive GPA at baseline, a higher S. aureus abundance was not associated with an increased relapse risk. Functional analyses identified ten SEED protein subsystems that differed between the groups. Most significant associations were related to chorismate synthesis and involved in the vitamin B12 pathway. Conclusion Our data revealed a distinct dysbiosis of the nasal microbiota in GPA patients compared with disease and healthy controls. Metagenomic sequencing demonstrated that this dysbiosis in active GPA patients is manifested by increased abundance of S. aureus and a depletion of S. epidermidis, further demonstrating the antagonist relationships between these species. SEED functional protein subsystem analysis identified an association between the unique bacterial nasal microbiota clusters seen mainly in GPA patients and an elevated abundance of genes associated with chorismate synthesis and vitamin B12 pathways. Further studies are required to further elucidate the relationship between the biosynthesis genes and the associated bacterial species.

Identification of Microbes Associated with the Urethra during Health and Inflammation

The number of cases of urethritis, or inflammation of the male urethra, in the US has been estimated to be 2.8 million each year in the United States.1 It is the most common reason young men seek primary care and this syndrome is associated with acute proctitis, epididymitis, and orchitis.2 Approximately half of the cases are idiopathic, meaning that a causative agent cannot be identified. The goal of my project is to identify and characterize novel microorganisms that may be associated with male idiopathic urethritis. We used a variety of approaches to cultivate microorganisms from archived urethral swabs collected from men with idiopathic urethritis and healthy controls. We defined the phylogeny of the isolates using 16S rRNA sequencing to identify organisms that are not presently represented in microbial databases. Currently, we are scaling up a number of novel taxa we identified for genome sequencing. By adding these new genome sequencing to the existing databases we will be able to assign a higher proportion of reads from corresponding metagenomic sequence data and achieve a more complete survey of the male urethral microbiota in health and urethritis. This will provide crucial information which may permit us to identify urethritis associated organisms, develop new molecular diagnostics for these organisms, and discern if how these organisms are sexually transmitted References 1. Brill JR. Diagnosis and treatment of urethritis in men. Am Fam Physician 2010; 81:873–8. 2. Bachmann LH, Manhart LE, Martin DH, Sena AC, Dimitrakoff J, Jensen JS, et al. Advances in the Understanding and Treatment of Male Urethritis. Clin Infect Dis. 2015;61 Suppl 8:S763–S9.

Improved metagenomic analysis with Kraken 2

Although Kraken’s k-mer-based approach provides fast taxonomic classification of metagenomic sequence data, its large memory requirements can be limiting for some applications. Kraken 2 improves upon Kraken 1 by reducing memory usage by 85%, allowing greater amounts of reference genomic data to be used, while maintaining high accuracy and increasing speed five-fold. Kraken 2 also introduces a translated search mode, providing increased sensitivity in viral metagenomics analysis.

The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from a cis-splicing intein

Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. The 1.0 Å-resolution crystal structure of a variant from naturally split gp41-1 intein, identified from the environmental metagenomic sequence data, revealed an improved pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N-and C-terminal intein fragments. We successfully created orthogonal split inteins by engineering a similar charge network in the same region of a cis-splicing intein. The same strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins.