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

Bertrand, Denis; Shaw, Jim; Kalathiyappan, Manesh; Ng, Amanda Hui Qi; Kumar, M. S. Binoj; Li, Chenhao; Dvorničić, Mirta; Soldo, Janja Paliska; Koh, Jia Yu; Tong, Chengxuan; Ng, Oon Tek; Barkham, Timothy; Young, Barnaby Edward; Marimuthu, Kalisvar; Chng, Kern Rei; Šikić, Mile; Nagarajan, Niranjan

doi:10.1038/s41587-019-0191-2

Cited by 244 publications

(262 citation statements)

References 70 publications

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“…Our results from these diverse and understudied groundwater ecosystems extend findings recently published that utilized mock communities and spiked-in complex human gut microbiome communities (Bertrand et al , 2019) . We demonstrate the recovery of a wider diversity of microorganisms and phages using a hybrid approach, in our case, the recovery of three additional phyla and 6 classes.…”

Section: Resultssupporting

confidence: 86%

“…One exciting avenue in reconstructing de novo MAGs has been the inclusion of long-read sequences that can act as scaffolds to short-read sequences to help improve contiguity and bridge repeat regions within a genome . This concept has been present since the advent of second-generation sequencing technologies (Roche 454, Illumina, SOLiD) (Goldberg et al , 2006) , but the breakthroughs in third-generation sequencing technologies, particularly PacBio and Oxford Nanopore Technologies (ONT), have improved the practicality of such approaches by providing access to much longer reads (Scholz et al , 2014;Frank et al , 2016;Bertrand et al , 2019) .…”

Section: Introductionmentioning

confidence: 99%

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Inclusion of Oxford Nanopore long reads improves all microbial and phage metagenome-assembled genomes from a complex aquifer system

Overholt¹,

Hölzer

Geesink³

et al. 2019

Preprint

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Assembling microbial and phage genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. In order to compare the recovery of genomes from microorganisms and their phages from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore sequencing platform. A ssembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from Illumina-only assemblies and a hybrid assembly approach. Strikingly, the hybrid approach more than doubled the number of mid to high-quality MAGs (> 50% completion, < 10% redundancy), generated nearly four-fold more phage genomes, and improved all associated genome metrics relative to

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Section: Resultssupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Inclusion of Oxford Nanopore long reads improves all microbial and phage metagenome-assembled genomes from a complex aquifer system

Overholt¹,

Hölzer

Geesink³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…challenges in the assembly process that need to be properly addressed (Nicholls et al, 2019). In particular, high error rates hinder the assembly of highly similar strains as the differences between the genomes can be indistinguishable from sequencing errors (Bertrand et al, 2019). Although several long-read assembly methods have been successfully used for single-genome de novo assembly Koren et al, 2017) and new promising tools are being developed for metagenomic assembly (Kolmogorov et al, 2019a), how well these methods perform with real metagenomic data still remains to be properly evaluated.…”

Section: Introductionmentioning

confidence: 99%

“…Although several long-read assembly methods have been successfully used for single-genome de novo assembly Koren et al, 2017) and new promising tools are being developed for metagenomic assembly (Kolmogorov et al, 2019a), how well these methods perform with real metagenomic data still remains to be properly evaluated. Yet, an increasing number of long read-based metagenomics studies demonstrate that it is possible to reconstruct complete or near-complete genomes from mock communities and natural microbial communities of varying complexity (Bertrand et al, 2019;Hao et al, 2018;Moss and Bhatt, 2018;Nicholls et al, 2019;Somerville et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Near-complete Lokiarchaeota genomes from complex environmental samples using long and short read metagenomic analyses

Caceres

Lewis

Homa

et al. 2019

Preprint

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Asgard archaea is a recently proposed superphylum currently comprised of five recognised phyla: Lokiarchaeota, Thorarchaeota, Odinarchaeota, Heimdallarchaeota and Helarchaeota. Members of this group have been identified based on culture-independent approaches with several metagenome-assembled genomes (MAGs) reconstructed to date. However, most of these genomes consist of several relatively small contigs, and, until recently, no complete Asgard archaea genome is yet available. Large scale phylogenetic analyses suggest that Asgard archaea represent the closest archaeal relatives of eukaryotes. In addition, members of this superphylum encode proteins that were originally thought to be specific to eukaryotes, including components of the trafficking machinery, cytoskeleton and endosomal sorting complexes required for transport (ESCRT). Yet, these findings have been questioned on the basis that the genome sequences that underpin them were assembled from metagenomic data, and could have been subjected to contamination and other assembly artefacts. Even though several lines of evidence indicate that the previously reported findings were not affected by these issues, having access to high-quality and preferentially fully closed Asgard archaea genomes is needed to definitively close this debate. Current long-read sequencing technologies such as Oxford Nanopore allow the generation of long reads in a high-throughput manner making them suitable for their use in metagenomics. Although the use of long reads is still limited in this field, recent analyses have shown that it is feasible to obtain complete or nearcomplete genomes of abundant members of mock communities and metagenomes of various level of complexity. Here, we show that long read metagenomics can be successfully applied to obtain near-complete genomes of low-abundant members of complex communities from sediment samples. We were able to reconstruct six MAGs from different Lokiarchaeota lineages that show high completeness and low fragmentation, with one of them being a nearcomplete genome only consisting of three contigs. Our analyses confirm that the eukaryote-like features previously associated with Lokiarchaeota are not the result of contamination or assembly artefacts, and can indeed be found in the newly reconstructed genomes.Recent advances in long-read DNA sequencing technologies have made long read-based metagenomic sequencing efforts a feasible option (Hao et al., 2018;Nicholls et al., 2019). Long DNA sequencing reads improve the ability to resolve repetitive regions of sequences in de novo assembly, improving the contiguity of genomic assemblies. In metagenomes, repetitive sequences can be present within a single genome, as well as within several genomes that share similar regions (e.g., closely related strains, horizontally transferred genes). Therefore, if reads are long enough to span across repetitive regions, and also cover regions that are unique to a particular genome, their inclusion in metagenome assemblies can help to separate the genomic sequenc...

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“…Hi-C read linkages and the overall proximity between Hi-C read linkages and the inferred transferred 243 genes (Extended Data Figure 7). Future studies should leverage long reads in hybrid assemblers to better 244 capture co-occurring AR genes and large MGEs 43 . We expect to overcome these limitations with 245…”

mentioning

confidence: 99%

Widespread transfer of mobile antibiotic resistance genes within individual gut microbiomes revealed through bacterial Hi-C

Kent

Vill

Shi

et al. 2020

Preprint

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12The gut microbiome harbors a 'silent reservoir' of antibiotic resistance (AR) genes that is thought to 13 contribute to the emergence of multidrug-resistant pathogens through the process of horizontal gene 14 transfer (HGT). To counteract the spread of AR genes, it is paramount to know which organisms harbor 15 mobile AR genes and with which organisms they engage in HGT. Despite methods to characterize the 16 bulk presence 1 , abundance 2 and function 3 of AR genes in the gut, technological limitations of short-read 17sequencing have precluded linking bacterial taxa to specific mobile genetic elements (MGEs) and their 18 concomitant AR genes. Here, we apply and evaluate a high-throughput, culture-independent method for 19surveilling the bacterial carriage of MGEs, based on bacterial Hi-C protocols. We compare two healthy 20individuals with a cohort of seven neutropenic patients undergoing hematopoietic stem cell 21 transplantation, who receive multiple courses of antibiotics throughout their prolonged hospitalizations, 22and are thus acutely vulnerable to the threat of multidrug-resistant infections 4 . We find that the networks 23 of HGT are surprisingly distinct between individuals, yet AR and mobile genes are more dispersed across 24 taxa within the neutropenic patients than the healthy subjects. Our data further suggest that HGT is 25occurring throughout the course of treatment in the microbiomes of neutropenic patients and within the 26 guts of healthy individuals over a similar timeframe. Whereas most efforts to understand the spread of AR 27genes have focused on pathogenic species, our findings shed light on the role of the human gut 28 microbiome in this process. 29 preferentially recruited to contigs that are longer and more abundant, but to a lesser degree than expected, 81reducing potential bias in our dataset toward highly abundant organisms (Extended Data Figure 4). We 82 binned contigs using several tools (Maxbin 22 , MetaBat and Concoct), and applied a binning aggregation 83 strategy, DAS Tool 23 , to obtain a set of draft genomic assemblies. As misassembly can resemble HGT, we 84removed assemblies with greater than 10% contamination, as determined by CheckM, resulting in 85 taxonomically coherent assemblies (Extended Data Figure 5), albeit a greater number of unbinned contigs 86(24.6% of the total) (Extended Data Table 3). We then apply conservative criteria to link mobile and 87 mobile AR-containing contigs with the genomic draft assemblies, considering an MGE part of a genome 88 assembly only if it is directly linked to it by at least two uniquely-mapped Hi-C read-pairs. As MGEs are 89 known to recombine, this mitigates the potential for falsely linking contigs that merely share common 90 mobile genes. However, this also potentially reduces our ability for overall detection, especially for larger 91MGEs, since mobile contigs are often fragmented in metagenomic assemblies 24 . Nevertheless, we 92restricted our analysis to those AR-organism and MGE-organism linkages derived from high-confidence ...

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

Cited by 244 publications

References 70 publications

Inclusion of Oxford Nanopore long reads improves all microbial and phage metagenome-assembled genomes from a complex aquifer system

Inclusion of Oxford Nanopore long reads improves all microbial and phage metagenome-assembled genomes from a complex aquifer system

Near-complete Lokiarchaeota genomes from complex environmental samples using long and short read metagenomic analyses

Widespread transfer of mobile antibiotic resistance genes within individual gut microbiomes revealed through bacterial Hi-C

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