GroopM: an automated tool for the recovery of population genomes from related metagenomes.
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ABSTRACT:
SUBMITTER: Imelfort M
PROVIDER: S-EPMC4183954 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Shotgun metagenomics is a powerful, high-resolution technique enabling the study of microbial communities in situ. However, species-level resolution is only achieved after a process of 'binning' where contigs predicted to originate from the same genome are clustered. Such culture-independent sequencing frequently unearths novel microbes, and so various methods have been devised for reference-free binning. As novel microbiomes of increasing complexity are explored, sometimes associated with non-model hosts, robust automated binning methods are required. Existing methods struggle with eukaryotic contamination and cannot handle highly complex single metagenomes. We therefore developed an automated binning pipeline, termed 'Autometa', to address these issues. This command-line application integrates sequence homology, nucleotide composition, coverage and the presence of single-copy marker genes to separate microbial genomes from non-model host genomes and other eukaryotic contaminants, before deconvoluting individual genomes from single metagenomes. The method is able to effectively separate over 1000 genomes from a metagenome, allowing the study of previously intractably complex environments at the level of single species. Autometa is freely available at https://bitbucket.org/jason_c_kwan/autometa and as a docker image at https://hub.docker.com/r/jasonkwan/autometa under the GNU Affero General Public License 3 (AGPL 3).
| S-EPMC6547426 | biostudies-literature
Project description:Microbial communities are critical to ecosystem function. A key objective of metagenomic studies is to analyse organism-specific metabolic pathways and reconstruct community interaction networks. This requires accurate assignment of assembled genome fragments to genomes. Existing binning methods often fail to reconstruct a reasonable number of genomes and report many bins of low quality and completeness. Furthermore, the performance of existing algorithms varies between samples and biotopes. Here, we present a dereplication, aggregation and scoring strategy, DAS Tool, that combines the strengths of a flexible set of established binning algorithms. DAS Tool applied to a constructed community generated more accurate bins than any automated method. Indeed, when applied to environmental and host-associated samples of different complexity, DAS Tool recovered substantially more near-complete genomes, including previously unreported lineages, than any single binning method alone. The ability to reconstruct many near-complete genomes from metagenomics data will greatly advance genome-centric analyses of ecosystems.
| S-EPMC6786971 | biostudies-literature
Project description:Long-read sequencing offers the potential to improve metagenome assemblies and provide more robust assessments of microbial community composition and function than short-read sequencing. We applied Pacific Biosciences (PacBio) CCS (circular consensus sequencing) HiFi shotgun sequencing to 14 marine water column samples and compared the results with those for short-read metagenomes from the corresponding environmental DNA samples. We found that long-read metagenomes varied widely in quality and biological information. The community compositions of the corresponding long- and short-read metagenomes were frequently dissimilar, suggesting higher stochasticity and/or bias associated with PacBio sequencing. Long reads provided few improvements to the assembly qualities, gene annotations, and prokaryotic metagenome-assembled genome (MAG) binning results. However, only long reads produced high-quality eukaryotic MAGs and contigs containing complete zooplankton marker gene sequences. These results suggest that high-quality long-read metagenomes can improve marine community composition analyses and provide important insight into eukaryotic phyto- and zooplankton genetics, but the benefits may be outweighed by the inconsistent data quality. IMPORTANCE Ocean microbes provide critical ecosystem services, but most remain uncultivated. Their communities can be studied through shotgun metagenomic sequencing and bioinformatic analyses, including binning draft microbial genomes. However, most sequencing to date has been done using short-read technology, which rarely yields genome sequences of key microbes like SAR11. Long-read sequencing can improve metagenome assemblies but is hampered by technological shortcomings and high costs. In this study, we compared long- and short-read sequencing of marine metagenomes. We found a wide range of long-read metagenome qualities and minimal improvements to microbiome analyses. However, long reads generated draft genomes of eukaryotic algal species and provided full-length marker gene sequences of zooplankton species, including krill and copepods. These results suggest that long-read sequencing can provide greater genetic insight into the wide diversity of eukaryotic phyto- and zooplankton that interact as part of and with the marine microbiome.
| S-EPMC9765425 | biostudies-literature
Project description:The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a 'dark matter.' We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (? 1 Terabase) were collected, processed, and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and 5 virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate 100s of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are effective in improving knowledge about the presence and prevalence of giant viruses in the environment and the human body.
| S-EPMC4814491 | biostudies-other
Project description:BackgroundRecovering individual genomes from metagenomic datasets allows access to uncultivated microbial populations that may have important roles in natural and engineered ecosystems. Understanding the roles of these uncultivated populations has broad application in ecology, evolution, biotechnology and medicine. Accurate binning of assembled metagenomic sequences is an essential step in recovering the genomes and understanding microbial functions.ResultsWe have developed a binning algorithm, MaxBin, which automates the binning of assembled metagenomic scaffolds using an expectation-maximization algorithm after the assembly of metagenomic sequencing reads. Binning of simulated metagenomic datasets demonstrated that MaxBin had high levels of accuracy in binning microbial genomes. MaxBin was used to recover genomes from metagenomic data obtained through the Human Microbiome Project, which demonstrated its ability to recover genomes from real metagenomic datasets with variable sequencing coverages. Application of MaxBin to metagenomes obtained from microbial consortia adapted to grow on cellulose allowed genomic analysis of new, uncultivated, cellulolytic bacterial populations, including an abundant myxobacterial population distantly related to Sorangium cellulosum that possessed a much smaller genome (5 MB versus 13 to 14 MB) but has a more extensive set of genes for biomass deconstruction. For the cellulolytic consortia, the MaxBin results were compared to binning using emergent self-organizing maps (ESOMs) and differential coverage binning, demonstrating that it performed comparably to these methods but had distinct advantages in automation, resolution of related genomes and sensitivity.ConclusionsThe automatic binning software that we developed successfully classifies assembled sequences in metagenomic datasets into recovered individual genomes. The isolation of dozens of species in cellulolytic microbial consortia, including a novel species of myxobacteria that has the smallest genome among all sequenced aerobic myxobacteria, was easily achieved using the binning software. This work demonstrates that the processes required for recovering genomes from assembled metagenomic datasets can be readily automated, an important advance in understanding the metabolic potential of microbes in natural environments. MaxBin is available at https://sourceforge.net/projects/maxbin/.
| S-EPMC4129434 | biostudies-literature
Project description:Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes.
| S-EPMC7111523 | biostudies-literature
Project description:Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.
| S-EPMC4270193 | biostudies-literature
Project description:Infectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.
| S-EPMC4589665 | biostudies-literature
Project description: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).
| S-EPMC3897386 | biostudies-literature
Project description:Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, few approaches exist for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identify nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. To refine bins, vRhyme utilizes unique features of viral genomes, namely a protein redundancy scoring mechanism based on the observation that viruses seldom encode redundant genes. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.
| S-EPMC9371927 | biostudies-literature