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Hybrid metagenomic assembly enables high-resolution analysis of resistance determinants and mobile elements in human microbiomes


Characterization of microbiomes has been enabled by high-throughput metagenomic sequencing. However, existing methods are not designed to combine reads from short- and long-read technologies. We present a hybrid metagenomic assembler named OPERA-MS that integrates assembly-based metagenome clustering with repeat-aware, exact scaffolding to accurately assemble complex communities. Evaluation using defined in vitro and virtual gut microbiomes revealed that OPERA-MS assembles metagenomes with greater base pair accuracy than long-read (>5×; Canu), higher contiguity than short-read (~10× NGA50; MEGAHIT, IDBA-UD, metaSPAdes) and fewer assembly errors than non-metagenomic hybrid assemblers (2×; hybridSPAdes). OPERA-MS provides strain-resolved assembly in the presence of multiple genomes of the same species, high-quality reference genomes for rare species (<1%) with ~9× long-read coverage and near-complete genomes with higher coverage. We used OPERA-MS to assemble 28 gut metagenomes of antibiotic-treated patients, and showed that the inclusion of long nanopore reads produces more contiguous assemblies (200× improvement over short-read assemblies), including more than 80 closed plasmid or phage sequences and a new 263 kbp jumbo phage. High-quality hybrid assemblies enable an exquisitely detailed view of the gut resistome in human patients.

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Fig. 1: OPERA-MS workflow.
Fig. 2: Benchmarking hybrid assembly of genomes from metagenomes.
Fig. 3: Assembly of a virtual gut microbiome.
Fig. 4: Mobile elements and association with host species in the human gut microbiome.

Data availability

GIS20 mock community sequencing data can be obtained from the European Nucleotide Archive (ENA) under project ID PRJEB29139 (Illumina, PacBio and ONT) and sequencing data for the 28 gut metagenomes can be found under project ID PRJEB29152 (Illumina and ONT).

Code availability

OPERA-MS is freely available under the MIT license at


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This work was supported by funding from the National Healthcare Group (NHG-CSCS/12008 and SIDI/2013/008) to K.M. and O.T.N., BMRC IAF (IAF311018) to K.M., O.T.N. and N.N., HBMS IAF-PP (H18/01/a0/016) and A*STAR Singapore to N.N.

Author information




D.B. and N.N. designed the algorithm with inputs from J.S. and M.S.K. D.B., J.S., M.K., M.S.K., M.D. and J.P.S. implemented OPERA-MS. D.B., M.K., C.L., J.Y.K., C.T. and K.R.C. conducted computational experiments and analysis with guidance from N.N. O.T.N., T.B., B.Y. and K.M. organized volunteer recruitment and sampling. A.H.Q.N. performed wet-lab experiments. D.B. and N.N. wrote the manuscript with inputs from M.K., K.R.C. and M.S. All authors read and approved the final manuscript.

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Correspondence to Niranjan Nagarajan.

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The authors declare no competing interests.

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Bertrand, D., Shaw, J., Kalathiyappan, M. et al. Hybrid metagenomic assembly enables high-resolution analysis of resistance determinants and mobile elements in human microbiomes. Nat Biotechnol 37, 937–944 (2019).

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