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Improved high-molecular-weight DNA extraction, nanopore sequencing and metagenomic assembly from the human gut microbiome


Short-read metagenomic sequencing and de novo genome assembly of the human gut microbiome can yield draft bacterial genomes without isolation and culture. However, bacterial genomes assembled from short-read sequencing are often fragmented. Furthermore, these metagenome-assembled genomes often exclude repeated genomic elements, such as mobile genetic elements, compromising our understanding of the contribution of these elements to important bacterial phenotypes. Although long-read sequencing has been applied successfully to the assembly of contiguous bacterial isolate genomes, extraction of DNA of sufficient molecular weight, purity and quantity for metagenomic sequencing from stool samples can be challenging. Here, we present a protocol for the extraction of microgram quantities of high-molecular-weight DNA from human stool samples that are suitable for downstream long-read sequencing applications. We also present Lathe (, a computational workflow for long-read basecalling, assembly, consensus refinement with long reads or Illumina short reads and genome circularization. Altogether, this protocol can yield high-quality contiguous or circular bacterial genomes from a complex human gut sample in approximately 10 d, with 2 d of hands-on bench and computational effort.

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Fig. 1: High-molecular-weight DNA extraction workflow.
Fig. 2: Post-sequencing bioinformatic workflow.

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Data availability

No new data were generated or analyzed for this manuscript.

Code availability

Lathe is available at Post-assembly binning workflows can be found at


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We thank all members of the Bhatt laboratory for experimental advice and discussions. We thank Brayon Fremin for making suggestions for the abbreviated DNA extraction protocol, and Matthew Grieshop, Keenan Manpearl, David Sanchez Godinez and Alexandra I. Strom for helpful comments on the manuscript. D.G.M. was supported by the Stanford Graduate Fellowships in Science and Engineering program. E.L.M. was supported by the National Science Foundation Graduate Research Fellowship no. DGE-114747. This work was supported by the Damon Runyon Clinical Investigator Award, grant nos. NIH R01AI148623 and NIH R01AI143757 to the Bhatt laboratory and grant no. NIH P30 AG047366, which supports the Stanford ADRC. Computational work was supported by NIH S10 Shared Instrumentation grant no. 1S10OD02014101 and by NIH grant no. P30 CA124435, which supports the Genetics Bioinformatics Service Center, a Stanford Cancer Institute Shared Resource.

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Authors and Affiliations



E.L.M. and A.S.B. conceived the study. E.L.M., D.G.M., S.E.V. and A.S.B. performed all experiments and data analysis. D.G.M., S.E.V. and A.S.B. wrote the paper with input from all authors. All authors read and approved the final manuscript.

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Correspondence to Ami S. Bhatt.

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The authors declare no competing interests.

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

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Moss, E. L., Maghini, D. G. & Bhatt, A. S. Nat. Biotechnol. 38, 701–707 (2020):

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Notes 1 and 2.

Reporting Summary

Supplementary Table 1

Summary of HMW DNA extraction methods

Supplementary Table 2

Lytic enzyme activity

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Maghini, D.G., Moss, E.L., Vance, S.E. et al. Improved high-molecular-weight DNA extraction, nanopore sequencing and metagenomic assembly from the human gut microbiome. Nat Protoc 16, 458–471 (2021).

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