Metaphor - A workflow for streamlined assembly and binning of metagenomes
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Abstract
Recent advances in bioinformatics and high-throughput sequencing have enabled the large-scale recovery of genomes from metagenomes. This has the potential to bring important insights as researchers can bypass cultivation and analyse genomes sourced directly from environmental samples. There are, however, technical challenges associated with this process, most notably the complexity of computational workflows required to process metagenomic data, which include dozens of bioinformatics software tools, each with their own set of customisable parameters that affect the final output of the workflow. At the core of these workflows are the processes of assembly - combining the short input reads into longer, contiguous fragments (contigs), and binning - clustering these contigs into individual genome bins. Both processes can be done for each sample separately or by pooling together multiple samples to leverage information from a combination of samples. Here we present Metaphor, a fully-automated workflow for genome-resolved metagenomics (GRM). Metaphor differs from existing GRM workflows by offering flexible approaches for the assembly and binning of the input data, and by combining multiple binning algorithms with a bin refinement step to achieve high quality genome bins. Moreover, Metaphor generates reports to evaluate the performance of the workflow. We showcase the functionality of Metaphor on different synthetic datasets, and the impact of available assembly and binning strategies on the final results. The workflow is freely available at https://github.com/vinisalazar/metaphor .
Author summary
We present Metaphor, a user-friendly, automated workflow for the recovery of genomes from metagenomes. Our tool offers flexible options for assembling and binning metagenomic contigs, that may be adjusted according to the characteristics of the input data and available computational resources, and a combination of binning algorithms, which improves the quantity and quality of resulting genome bins. We showcase the performance of Metaphor on synthetic benchmarking datasets and discuss the implication of methodological decisions regarding the strategy for assembling and binning metagenomic contigs.
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AbstractRecent advances in bioinformatics and high-throughput sequencing have enabled the large-scale recovery of genomes from metagenomes. This has the potential to bring important insights as researchers can bypass cultivation and analyse genomes sourced directly from environmental samples. There are, however, technical challenges associated with this process, most notably the complexity of computational workflows required to process metagenomic data, which include dozens of bioinformatics software tools, each with their own set of customisable parameters that affect the final output of the workflow. At the core of these workflows are the processes of assembly - combining the short input reads into longer, contiguous fragments (contigs), and binning - clustering these contigs into individual genome bins. Both processes can be done …
AbstractRecent advances in bioinformatics and high-throughput sequencing have enabled the large-scale recovery of genomes from metagenomes. This has the potential to bring important insights as researchers can bypass cultivation and analyse genomes sourced directly from environmental samples. There are, however, technical challenges associated with this process, most notably the complexity of computational workflows required to process metagenomic data, which include dozens of bioinformatics software tools, each with their own set of customisable parameters that affect the final output of the workflow. At the core of these workflows are the processes of assembly - combining the short input reads into longer, contiguous fragments (contigs), and binning - clustering these contigs into individual genome bins. Both processes can be done for each sample separately or by pooling together multiple samples to leverage information from a combination of samples. Here we present Metaphor, a fully-automated workflow for genome-resolved metagenomics (GRM). Metaphor differs from existing GRM workflows by offering flexible approaches for the assembly and binning of the input data, and by combining multiple binning algorithms with a bin refinement step to achieve high quality genome bins. Moreover, Metaphor generates reports to evaluate the performance of the workflow. We showcase the functionality of Metaphor on different synthetic datasets, and the impact of available assembly and binning strategies on the final results. The workflow is freely available at https://github.com/vinisalazar/metaphor.Author summary
**Reviewer 2. Po-Yu Liu **
The Metaphor is a workflow with high completeness for short-read-based metagenomic analysis. I look forward to its compatibility with long-read platforms (ONT and PacBio). This work is worth publishing. However, it is still a bioinformatic knowledge and skill-required toolkit. If the Metaphor can be integrated into a web-based platform, such as Galaxy or Kbase, it would be more user-friendly for much more users.
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AbstractRecent advances in bioinformatics and high-throughput sequencing have enabled the large-scale recovery of genomes from metagenomes. This has the potential to bring important insights as researchers can bypass cultivation and analyse genomes sourced directly from environmental samples. There are, however, technical challenges associated with this process, most notably the complexity of computational workflows required to process metagenomic data, which include dozens of bioinformatics software tools, each with their own set of customisable parameters that affect the final output of the workflow. At the core of these workflows are the processes of assembly - combining the short input reads into longer, contiguous fragments (contigs), and binning - clustering these contigs into individual genome bins. Both processes can be done …
AbstractRecent advances in bioinformatics and high-throughput sequencing have enabled the large-scale recovery of genomes from metagenomes. This has the potential to bring important insights as researchers can bypass cultivation and analyse genomes sourced directly from environmental samples. There are, however, technical challenges associated with this process, most notably the complexity of computational workflows required to process metagenomic data, which include dozens of bioinformatics software tools, each with their own set of customisable parameters that affect the final output of the workflow. At the core of these workflows are the processes of assembly - combining the short input reads into longer, contiguous fragments (contigs), and binning - clustering these contigs into individual genome bins. Both processes can be done for each sample separately or by pooling together multiple samples to leverage information from a combination of samples. Here we present Metaphor, a fully-automated workflow for genome-resolved metagenomics (GRM). Metaphor differs from existing GRM workflows by offering flexible approaches for the assembly and binning of the input data, and by combining multiple binning algorithms with a bin refinement step to achieve high quality genome bins. Moreover, Metaphor generates reports to evaluate the performance of the workflow. We showcase the functionality of Metaphor on different synthetic datasets, and the impact of available assembly and binning strategies on the final results. The workflow is freely available at https://github.com/vinisalazar/metaphor.
This work has been published in GigaByte Journal under a CC-BY 4.0 license (https://doi.org/10.1093/gigascience/giad055) and has published the reviews under the same license. These are as follows.
**Reviewer 1. Thomas Brüls **
The authors present a snakemake-based workflow to automate and chain the main computational ingredients (assembly and binning) of genome-centric metagenomics; the authors developed a technically sound tool for this purpose, and by itself it is certainly valuable to the research community and worth of publication. however, even if the article is casted as a technical note -hence with an emphasis on the design, implementation and assessment of the tool-, I feel that a more thorough discussion of both its abilities and inabilities (e.g. strain resolution, detection of low abundance organisms, identification of virus bins, etc) would be worth for a more general audience. On the same token, a more deep discussion of some of the results obtained with their tool (see below) would be of interest and would also illustrate useful use cases. I would suggest the following modifications/additions:-the experiments with the strain madness dataset suggest that the genomes (or fragments thereof, i.e. the bins) resolved should be viewed as "species" genomes, or composite genomes possibly originating from multiple strains. if so, do the authors think this represents a hard limit to the assembly + binning approach, or could further existing tools (e.g. performing variant detection on top of cross-assembly before the binning step) be integrated or developed in the future for strain-resolution (i.e. to identify strains not dominant in any sample)? -related, a simple summary of the number of individual strains recovered in individual bins for the strain madness experiment would be interesting.-another issue that would be worth discussing in my opinion is the impact of genome abundance on the recovery of corresponding bins and their quality. the platform developed by the authors appears to be well suited for such kind of analyses and the results would be of both theoretical and practical interest. to put it simply, what is the minimal initial coverage of genomes required in order for them to be recovered in bins of a given size and quality?-rem: theses two issues (strain-level diversity and individual strain genome abundances) likely interact to limit bin resolution, and this could be mentioned by the authors.-the data presented by the authors suggest that the metabat binning engine significantly outperforms the other two tools (concoct and vamb, which are both widely used), see e.g Figure 2; what would account for that, and do the authors think this is a general observation (i.e. beyond the specific CACB setting or marine metagenome shown in Fig 2)? -a bin refinement step (based on the DAS tool and dereplication) is frequently mentioned but should be more detailed (including a precise definition of the bin quality metric used).
further rather minor comments: -in the abstract, when mentioning "technical challenges associated with...", it would be worth mentioning that algorithmic challenges are present as well. -in the introduction, "It is hypothesised that pooled assembly and binning may lead to improved results when analysing communities with high genetic diversity, and to poorer results when there is a high level of intraspecies/strain-level diversity". I would assume there are many instances in the real world that are both, i.e. that present both high inter-species and intra-species genetic diversity, what then?-in the future directions, the authors mention the identification of eukaryotic and viral contigs and bins, and could shortly elaborate how this could be done properly. -the sentence "In summary, our assessment of ..." at the end of the ms appears to have a syntactic problem.
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