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Recovering prokaryotic genomes from host-associated, short-read shotgun metagenomic sequencing data

Nature Protocols volume 16, pages 2520–2541 (2021)Cite this article

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Abstract

Recovering genomes from shotgun metagenomic sequence data allows detailed taxonomic and functional characterization of individual species or strains in a microbial community. Retrieving these metagenome-assembled genomes (MAGs) involves seven stages. First, low-quality bases, along with adapter and host sequences, are removed. Second, overlapping sequences are assembled to create longer contiguous fragments. Third, these fragments are clustered based on sequence composition and abundance. Fourth, these sequence clusters, or bins, undergo rounds of quality assessment and refinement to yield MAGs. The optional fifth stage is dereplication of MAGs to select representatives. Next, each MAG is taxonomically classified. The optional seventh stage is assessing the fraction of diversity that has been recovered. The output of this protocol is draft genomes, which can provide invaluable clues about uncultured organisms. This protocol takes ~1 week to run, depending on computational resources available, and requires prior experience with high-performance computing, shell script programming and Python.

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Fig. 1: Overview of workflow for recovery of prokaryotic MAGs from shotgun metagenomic sequence data.
Fig. 2: MULTIQC plots of metagenomic sequence datasets after preprocessing.
Fig. 3: Quality assessment and taxonomic classification of prokaryotic MAGs.
Fig. 4: Using read mapping to visualize bottlenecks in assembly and binning.

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Data availability

The data presented in Figs. 2 and 3 are available in the supporting primary research papers. The raw data files for the gut runs in Figs. 2–4 are available as part of the project accessions PRJNA268964 and PRJNA278393. The raw data files for the skin runs in Fig. 4 are available via the project accession PRJNA46333. The datasets generated for this protocol in Figs. 2–4 are available in https://github.com/Finn-Lab/MAG_Snakemake_wf under the subfolder Anticipated_Results.

Code availability

Codes used in this protocol are publicly available at https://github.com/Finn-Lab/MAG_Snakemake_wf. The code in this protocol has been peer reviewed.

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Acknowledgements

S.S.K. is a graduate student supported by the NIH-Oxford-Cambridge Scholars Program. A.A. and R.D.F. are funded by EMBL core funds.

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Authors and Affiliations

  1. European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, UK

    Sara Saheb Kashaf, Alexandre Almeida & Robert D. Finn

  2. Microbial Genomics Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

    Sara Saheb Kashaf & Julia A. Segre

  3. Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK

    Alexandre Almeida

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Contributions

S.S.K, A.A. and R.D.F. conceived the study. S.S.K. and A.A. wrote the pipeline and performed the analyses. A.A., J.A.S. and R.D.F. supervised the work. J.A.S. and R.D.F. provided funding. S.S.K., A.A. and R.D.F wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Robert D. Finn.

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Peer review information Nature Protocols thanks Matthew Olm, Maria Pachiadaki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Almeida, A. et al. Nature 568, 499–504 (2019): https://doi.org/10.1038/s41586-019-0965-1

Almeida, A. et al. Nat. Biotechnol. 39, 105–114 (2021): https://doi.org/10.1038/s41587-020-0603-3

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Saheb Kashaf, S., Almeida, A., Segre, J.A. et al. Recovering prokaryotic genomes from host-associated, short-read shotgun metagenomic sequencing data. Nat Protoc 16, 2520–2541 (2021). https://doi.org/10.1038/s41596-021-00508-2

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