Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

Wick, Ryan R.; Judd, Louise M.; Holt, Kathryn E.

doi:10.1371/journal.pcbi.1010905

Cited by 70 publications

(44 citation statements)

References 54 publications

(67 reference statements)

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“…The cost per Gbp of Nanopore is currently between Illumina and PacBio. Nanopore technologies offer the affordability and benefits of recovering longer reads or the possibility of including short technologies for the better recovery of high-quality MAGs [ 4 , 10 , 50 ]. Nonetheless, sequencing error and read lengths should be considered when selecting between LR technologies [ 4 ].…”

Section: Resultsmentioning

confidence: 99%

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

et al. 2023

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Background Over the past years, sequencing technologies have expanded our ability to examine novel microbial metabolisms and diversity previously obscured by isolation approaches. Long-read sequencing promises to revolutionize the metagenomic field and recover less fragmented genomes from environmental samples. Nonetheless, how to best benefit from long-read sequencing and whether long-read sequencing can provide recovered genomes of similar characteristics as short-read approaches remains unclear. Results We recovered metagenome-assembled genomes (MAGs) from the free-living fraction at four-time points during a spring bloom in the North Sea. The taxonomic composition of all MAGs recovered was comparable between technologies. However, differences consisted of higher sequencing depth for contigs and higher genome population diversity in short-read compared to long-read metagenomes. When pairing population genomes recovered from both sequencing approaches that shared ≥ 99% average nucleotide identity, long-read MAGs were composed of fewer contigs, a higher N50, and a higher number of predicted genes when compared to short-read MAGs. Moreover, 88% of the total long-read MAGs carried a 16S rRNA gene compared to only 23% of MAGs recovered from short-read metagenomes. Relative abundances for population genomes recovered using both technologies were similar, although disagreements were observed for high and low GC content MAGs. Conclusions Our results highlight that short-read technologies recovered more MAGs and a higher number of species than long-read due to an overall higher sequencing depth. Long-read samples produced higher quality MAGs and similar species composition compared to short-read sequencing. Differences in the GC content recovered by each sequencing technology resulted in divergences in the diversity recovered and relative abundance of MAGs within the GC content boundaries.

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Section: Resultsmentioning

confidence: 99%

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

et al. 2023

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“…Hybrid assemblies incorporating short-and long-read data were created using Unicycler v.0.4.08 with standard parameters 41 with Unicycler output used to assess circularisation. If blaIMP-4 contigs were noncircularised, we re-assembled genomes using a long-read-first assembly using a bespoke pipeline (https://github.com/HughCottingham/clinopore-nf) that incorporates Flye with subsequent polishing with Medaka, Polypolish and Polca [42][43][44][45] . Assembly quality was checked using Quast 46 and species identification was performed using GTDB-Tk 47 and checked against isolate identification performed at time of isolate collection.…”

Section: De Novo Assembly and Annotationmentioning

confidence: 99%

Genomic dissection of endemic carbapenem resistance: metallo-beta-lactamase gene dissemination through clonal, plasmid and integron transfer pathways

Macesic

Hawkey

Vezina

et al. 2023

Preprint

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Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish endemicity remains limited. We analysed two decades of blaIMP-4 evolution in a hospital using sequence data from 270 clinical and environmental isolates (including 169 completed genomes) and identified extreme gene promiscuity across 7 Gram-negative genera, 68 bacterial strains and 7 distinct plasmid types. An initial multi-species outbreak of conserved IncC plasmids (95 genomes across 37 strains) allowed endemicity to be established through the ability of blaIMP-4 to disseminate in successful strain-genetic setting pairs we termed propagators, in particular Serratia marcescens and Enterobacter hormaechei. From this reservoir, blaIMP-4 persisted through diversification of genetic settings that resulted from transfer of blaIMP-4 plasmids between bacterial hosts and of the integron carrying blaIMP-4 between plasmids. Our findings provide a framework for understanding endemicity and spread of MBLs and may have broader applicability to other carbapenemase-producing organisms.

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“…Here, ten novel T7-like phages belonging to the genus Przondovirus in the family Autographiviridae have been isolated against four Klebsiella strains belonging to different species, and characterised. Hybrid poly-polish assembly methods have recently been described for assembling bacterial genomes [24]. We developed and validated a similar approach to ensure accurate and complete phage genome assembly, in a new worklow HYPPA, a HY brid and P oly-polish P hage A ssembly, which was tested and validated for these new phages.…”

Section: Introductionmentioning

confidence: 99%

A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses

et al. 2023

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Bacteriophages (phages) within the genus Przondovirus are T7-like podoviruses belonging to the subfamily Studiervirinae, within the family Autographiviridae, and have a highly conserved genome organisation. The genomes of these phages range from 37 to 42 kb in size, encode 50–60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA, a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our assembly workflow utilised Oxford Nanopore Technologies for long-read sequencing for its accessibility, making it the more relevant long-read sequencing technology at this time, and Illumina DNA Prep for short-read sequencing, representing the most commonly used technologies globally. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics.

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Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

Cited by 70 publications

References 54 publications

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

Genomic dissection of endemic carbapenem resistance: metallo-beta-lactamase gene dissemination through clonal, plasmid and integron transfer pathways

A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses

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