Analysis Of Metagenomic Sequencing Data Research Articles

New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp.

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

Helicobacter spp. are prevalent in wild mice and protect from lethal Citrobacter rodentium infection in the absence of adaptive immunity

Transfer of the gut microbiota from wild to laboratory mice alters the host's immune status and enhances resistance to infectious and metabolic diseases, but understanding of which microbes and how they promote host fitness is only emerging. Our analysis of metagenomic sequencing data reveals that Helicobacter spp. are enriched in wild compared with specific-pathogen-free (SPF) and conventionally housed mice, with multiple species commonly co-colonizing their hosts. We create laboratory mice harboring three non-SPF Helicobacter spp. to evaluate their effect on mucosal immunity and colonization resistance to the enteropathogen Citrobacter rodentium. Our experiments reveal that Helicobacter spp. interfere with C.rodentium colonization and attenuate C.rodentium-induced gut inflammation in wild-type (WT) mice, even preventing lethal infection in Rag2-/- SPF mice. Further analyses suggest that Helicobacter spp. interfere with tissue attachment of C.rodentium, putatively by reducing the availability of mucus-derived sugars. These results unveil pivotal protective functions of wild mouse microbiota constituents against intestinal infection.

MetaPrism: A versatile toolkit for joint taxa/gene analysis of metagenomic sequencing data.

In microbiome research, metagenomic sequencing generates enormous amounts of data. These data are typically classified into taxa for taxonomy analysis, or into genes for functional analysis. However, a joint analysis where the reads are classified into taxa-specific genes is often overlooked. To enable the analysis of this biologically meaningful feature, we developed a novel bioinformatic toolkit, MetaPrism, which can analyze sequence reads for a set of joint taxa/gene analyses to: 1) classify sequence reads and estimate the abundances for taxa-specific genes; 2) tabularize and visualize taxa-specific gene abundances; 3) compare the abundances between groups; and 4) build prediction models for clinical outcome. We illustrated these functions using a published microbiome metagenomics dataset from patients treated with immune checkpoint inhibitor therapy and showed the joint features can serve as potential biomarkers to predict therapeutic responses. MetaPrism is a toolkit for joint taxa and gene analysis. It offers biological insights on the taxa-specific genes on top of the taxa-alone or gene-alone analysis.MetaPrism is open-source software and freely available at https://github.com/jiwoongbio/MetaPrism. The example script to reproduce the manuscript is also provided in the above code repository.

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.

High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing.

The rise of antimicrobial-resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here, we show an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20× reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.

PSORTm: a bacterial and archaeal protein subcellular localization prediction tool for metagenomics data

MotivationMany methods for microbial protein subcellular localization (SCL) prediction exist; however, none is readily available for analysis of metagenomic sequence data, despite growing interest from researchers studying microbial communities in humans, agri-food relevant organisms and in other environments (e.g. for identification of cell-surface biomarkers for rapid protein-based diagnostic tests). We wished to also identify new markers of water quality from freshwater samples collected from pristine versus pollution-impacted watersheds.ResultsWe report PSORTm, the first bioinformatics tool designed for prediction of diverse bacterial and archaeal protein SCL from metagenomics data. PSORTm incorporates components of PSORTb, one of the most precise and widely used protein SCL predictors, with an automated classification by cell envelope. An evaluation using 5-fold cross-validation with in silico-fragmented sequences with known localization showed that PSORTm maintains PSORTb’s high precision, while sensitivity increases proportionately with metagenomic sequence fragment length. PSORTm’s read-based analysis was similar to PSORTb-based analysis of metagenome-assembled genomes (MAGs); however, the latter requires non-trivial manual classification of each MAG by cell envelope, and cannot make use of unassembled sequences. Analysis of the watershed samples revealed the importance of normalization and identified potential biomarkers of water quality. This method should be useful for examining a wide range of microbial communities, including human microbiomes, and other microbiomes of medical, environmental or industrial importance.Availability and implementationDocumentation, source code and docker containers are available for running PSORTm locally at https://www.psort.org/psortm/ (freely available, open-source software under GNU General Public License Version 3).Supplementary information Supplementary data are available at Bioinformatics online.

CoreProbe: A Novel Algorithm for Estimating Relative Abundance Based on Metagenomic Reads.

With the rapid development of high-throughput sequencing technology, the analysis of metagenomic sequencing data and the accurate and efficient estimation of relative microbial abundance have become important ways to explore the microbial composition and function of microbes. In addition, the accuracy and efficiency of the relative microbial abundance estimation are closely related to the algorithm and the selection of the reference sequence for sequence alignment. We introduced the microbial core genome as the reference sequence for potential microbes in a metagenomic sample, and we constructed a finite mixture and latent Dirichlet models and used the Gibbs sampling algorithm to estimate the relative abundance of microorganisms. The simulation results showed that our approach can improve the efficiency while maintaining high accuracy and is more suitable for high-throughput metagenomic data. The new approach was implemented in our CoreProbe package which provides a pipeline for an accurate and efficient estimation of the relative abundance of microbes in a community. This tool is available free of charge from the CoreProbe’s website: Access the Docker image with the following instruction: sudo docker pull panhongfei/coreprobe:1.0.

Measuring Cluster Stability in a Large Scale Phylogenetic Analysis of Functional Genes in Metagenomes Using pplacer.

Analysis of metagenomic sequence data requires a multi-stage workflow. The results of each intermediate step possess an inherent uncertainty and potentially impact the as-yet-unmeasured statistical significance of downstream analyses. Here, we describe our phylogenetic analysis pipeline which uses the pplacer program to place many shotgun sequences corresponding to a single functional gene onto a fixed phylogenetic tree. We then use the squash clustering method to compare multiple samples with respect to that gene. We approximate the statistical significance of each gene's clustering result by measuring its cluster stability, the consistency of that clustering result when the probabilistic placements made by pplacer are systematically reassigned and then clustered again, as measured by the adjusted Rand Index. We find that among the genes investigated, the majority of analyses are stable, based on the average adjusted Rand Index. We investigated properties of each gene that may explain less stable results. These genes tended to have less convex reference trees, less total reads recruited to the gene, and a greater Expected Distance between Placement Locations as given by pplacer when examined in aggregate. However, for an individual functional gene, these measures alone do not predict cluster stability.

MG-RAST, a Metagenomics Service for Analysis of Microbial Community Structure and Function.

Approaches in molecular biology, particularly those that deal with high-throughput sequencing of entire microbial communities (the field of metagenomics), are rapidly advancing our understanding of the composition and functional content of microbial communities involved in climate change, environmental pollution, human health, biotechnology, etc. Metagenomics provides researchers with the most complete picture of the taxonomic (i.e., what organisms are there) and functional (i.e., what are those organisms doing) composition of natively sampled microbial communities, making it possible to perform investigations that include organisms that were previously intractable to laboratory-controlled culturing; currently, these constitute the vast majority of all microbes on the planet. All organisms contained in environmental samples are sequenced in a culture-independent manner, most often with 16S ribosomal amplicon methods to investigate the taxonomic or whole-genome shotgun-based methods to investigate the functional content of sampled communities. Metagenomics allows researchers to characterize the community composition and functional content of microbial communities, but it cannot show which functional processes are active; however, near parallel developments in transcriptomics promise a dramatic increase in our knowledge in this area as well. Since 2008, MG-RAST (Meyer et al., BMC Bioinformatics 9:386, 2008) has served as a public resource for annotation and analysis of metagenomic sequence data, providing a repository that currently houses more than 150,000 data sets (containing 60+ tera-base-pairs) with more than 23,000 publically available. MG-RAST, or the metagenomics RAST (rapid annotation using subsystems technology) server makes it possible for users to upload raw metagenomic sequence data in (preferably) fastq or fasta format. Assessments of sequence quality, annotation with respect to multiple reference databases, are performed automatically with minimal input from the user (see Subheading 4 at the end of this chapter for more details). Post-annotation analysis and visualization are also possible, directly through the web interface, or with tools like matR (metagenomic analysis tools for R, covered later in this chapter) that utilize the MG-RAST API ( http://api.metagenomics.anl.gov/api.html ) to easily download data from any stage in the MG-RAST processing pipeline. Over the years, MG-RAST has undergone substantial revisions to keep pace with the dramatic growth in the number, size, and types of sequence data that accompany constantly evolving developments in metagenomics and related -omic sciences (e.g., metatranscriptomics).

Correction: TreeSeq, a Fast and Intuitive Tool for Analysis of Whole Genome and Metagenomic Sequence Data.

[This corrects the article DOI: 10.1371/journal.pone.0123851.].

VizBin - an application for reference-independent visualization and human-augmented binning of metagenomic data.

BackgroundMetagenomics is limited in its ability to link distinct microbial populations to genetic potential due to a current lack of representative isolate genome sequences. Reference-independent approaches, which exploit for example inherent genomic signatures for the clustering of metagenomic fragments (binning), offer the prospect to resolve and reconstruct population-level genomic complements without the need for prior knowledge.ResultsWe present VizBin, a Java™-based application which offers efficient and intuitive reference-independent visualization of metagenomic datasets from single samples for subsequent human-in-the-loop inspection and binning. The method is based on nonlinear dimension reduction of genomic signatures and exploits the superior pattern recognition capabilities of the human eye-brain system for cluster identification and delineation. We demonstrate the general applicability of VizBin for the analysis of metagenomic sequence data by presenting results from two cellulolytic microbial communities and one human-borne microbial consortium. The superior performance of our application compared to other analogous metagenomic visualization and binning methods is also presented.ConclusionsVizBin can be applied de novo for the visualization and subsequent binning of metagenomic datasets from single samples, and it can be used for the post hoc inspection and refinement of automatically generated bins. Due to its computational efficiency, it can be run on common desktop machines and enables the analysis of complex metagenomic datasets in a matter of minutes. The software implementation is available at https://claczny.github.io/VizBin under the BSD License (four-clause) and runs under Microsoft Windows™, Apple Mac OS X™ (10.7 to 10.10), and Linux.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-014-0066-1) contains supplementary material, which is available to authorized users.

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using 14C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of 14C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)'s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.

PhyloSift: phylogenetic analysis of genomes and metagenomes

Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection.In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata.These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454).

Functional assignment of metagenomic data: challenges and applications.

Metagenomic sequencing provides a unique opportunity to explore earth’s limitless environments harboring scores of yet unknown and mostly unculturable microbes and other organisms. Functional analysis of the metagenomic data plays a central role in projects aiming to explore the most essential questions in microbiology, namely ‘In a given environment, among the microbes present, what are they doing, and how are they doing it?’ Toward this goal, several large-scale metagenomic projects have recently been conducted or are currently underway. Functional analysis of metagenomic data mainly suffers from the vast amount of data generated in these projects. The shear amount of data requires much computational time and storage space. These problems are compounded by other factors potentially affecting the functional analysis, including, sample preparation, sequencing method and average genome size of the metagenomic samples. In addition, the read-lengths generated during sequencing influence sequence assembly, gene prediction and subsequently the functional analysis. The level of confidence for functional predictions increases with increasing read-length. Usually, the most reliable functional annotations for metagenomic sequences are achieved using homology-based approaches against publicly available reference sequence databases. Here, we present an overview of the current state of functional analysis of metagenomic sequence data, bottlenecks frequently encountered and possible solutions in light of currently available resources and tools. Finally, we provide some examples of applications from recent metagenomic studies which have been successfully conducted in spite of the known difficulties.

MicrobesOnline: an integrated portal for comparative and functional genomics

Since 2003, MicrobesOnline (http://www.microbesonline.org) has been providing a community resource for comparative and functional genome analysis. The portal includes over 1000 complete genomes of bacteria, archaea and fungi and thousands of expression microarrays from diverse organisms ranging from model organisms such as Escherichia coli and Saccharomyces cerevisiae to environmental microbes such as Desulfovibrio vulgaris and Shewanella oneidensis. To assist in annotating genes and in reconstructing their evolutionary history, MicrobesOnline includes a comparative genome browser based on phylogenetic trees for every gene family as well as a species tree. To identify co-regulated genes, MicrobesOnline can search for genes based on their expression profile, and provides tools for identifying regulatory motifs and seeing if they are conserved. MicrobesOnline also includes fast phylogenetic profile searches, comparative views of metabolic pathways, operon predictions, a workbench for sequence analysis and integration with RegTransBase and other microbial genome resources. The next update of MicrobesOnline will contain significant new functionality, including comparative analysis of metagenomic sequence data. Programmatic access to the database, along with source code and documentation, is available at http://microbesonline.org/programmers.html.

XplorSeq: a software environment for integrated management and phylogenetic analysis of metagenomic sequence data.

BackgroundAdvances in automated DNA sequencing technology have accelerated the generation of metagenomic DNA sequences, especially environmental ribosomal RNA gene (rDNA) sequences. As the scale of rDNA-based studies of microbial ecology has expanded, need has arisen for software that is capable of managing, annotating, and analyzing the plethora of diverse data accumulated in these projects.ResultsXplorSeq is a software package that facilitates the compilation, management and phylogenetic analysis of DNA sequences. XplorSeq was developed for, but is not limited to, high-throughput analysis of environmental rRNA gene sequences. XplorSeq integrates and extends several commonly used UNIX-based analysis tools by use of a Macintosh OS-X-based graphical user interface (GUI). Through this GUI, users may perform basic sequence import and assembly steps (base-calling, vector/primer trimming, contig assembly), perform BLAST (Basic Local Alignment and Search Tool; [1-3]) searches of NCBI and local databases, create multiple sequence alignments, build phylogenetic trees, assemble Operational Taxonomic Units, estimate biodiversity indices, and summarize data in a variety of formats. Furthermore, sequences may be annotated with user-specified meta-data, which then can be used to sort data and organize analyses and reports. A document-based architecture permits parallel analysis of sequence data from multiple clones or amplicons, with sequences and other data stored in a single file.ConclusionXplorSeq should benefit researchers who are engaged in analyses of environmental sequence data, especially those with little experience using bioinformatics software. Although XplorSeq was developed for management of rDNA sequence data, it can be applied to most any sequencing project. The application is available free of charge for non-commercial use at .