Abstract
Third-generation long-read sequencing technologies, significantly improve metagenome assemblies. Highly accurate PacBio HiFi reads can yield hundreds of near-complete metagenome-assembled genomes (MAGs) from a single sample. Recently, the accuracy of the more cost-effective Oxford Nanopore Technologies (ONT) platform has increased to a per-base error rate of 1-2%. However, current metagenome assemblers are optimized for HiFi and do not scale to the large data sets that ONT enables. We present nanoMDBG, an evolution of metaMDBG, which supports the latest ONT reads through an error correction pre-processing step in minimizer-space. Across a range of ONT datasets, including a large 400 Gbp soil sample, nanoMDBG reconstructs up to twice as many high-quality MAGs as the next best ONT assembler, metaFlye, while requiring a third of the CPU time and memory. Critically, the latest ONT technology can now produce comparable MAG construction results as those obtained using PacBio HiFi at the same sequencing depth.
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Data availability
The sequence data generated in this study have been deposited in the European Nucleotide Archive as the BioProject PRJEB88618. The individual accession numbers of all sequences used are: ERR15316007: Zymo ONT; ERR15285694: Human gut ONT; ERR15289757: Soil ONT; ERR15289675: Human gut HiFi; ERR15289804: Soil HiFi. Zymo mock reference genomes are available at https://s3.amazonaws.com/zymo-files/BioPool/D6331.refseq.zip. The ONT Zymo Fecal Reference data set is available at https://epi2me.nanoporetech.com/lc2024-datasets/. The HiFi Zymo Fecal Reference data set is available at https://www.pacb.com/connect/datasets/#metagenomics-datasets. Source data are provided with this paper.
Code availability
We implemented the nanoMDBG method in the metaMDBG software (https://github.com/GaetanBenoitDev/metaMDBG). The nanopore mode is activated using the input parameter (–in-ont), and the original PacBio HiFi mode using the parameter (–in-hifi). The analysis scripts used in this study to compare assemblers are available at https://github.com/GaetanBenoitDev/NanoMDBG_Manuscript.
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Acknowledgements
C.Q. and S.R. acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC), part of UK Research and Innovation; Earlham Institute Strategic Program (ISP) Grant (Decoding Biodiversity) BBX011089/1 and its constituent work package BBS/E/ER/230002C; the Core Strategic Program Grant (Genomes to Food Security) BB/CSP1720/1 and its constituent work packages BBS/E/T/000PR9818 and BBS/E/T/000PR9817; and the Core Capability Grant BB/CCG2220/1. C.Q. and R.J. acknowledge the QIB Food Microbiome and Health ISP BB/X011054/1 and its constituent project BBS/E/F/000PR13631. The authors gratefully acknowledge the support of the QIB Colon Model Facility, which was funded by the BBSRC Core Capability Grant BB/CCG2260/1. R.C. was supported by ANR grants ANR-22-CE45-0007, ANR-19-CE45-0008, PIA/ANR16-CONV-0005, ANR-19-P3IA-0001, ANR-21-CE46-0012-03, and Horizon Europe grants No. 872539, 956229, 101047160 and 101088572 (ERC IndexThePlanet, also supporting G.B.). We acknowledge the assistance of Dr. Susheel Bhanu Busi (CEH, Wallingford) in organizing the soil sampling.
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G.B. devised and implemented the approach and performed analysis with assistance from S.R., R.J., and G.A. prepared DNA extracts for sequencing and constructed libraries. T.G. collected soil samples. G.B., R.C., and C.Q. conceived the study and supervised and coordinated the work. All authors wrote, reviewed, edited and approved the manuscript.
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Benoit, G., James, R., Raguideau, S. et al. High-quality metagenome assembly from nanopore reads with nanoMDBG. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69760-y
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DOI: https://doi.org/10.1038/s41467-026-69760-y
