Polishing De Novo Nanopore Assemblies of Bacteria and Eukaryotes With FMLRC2.

As the accuracy and throughput of nanopore sequencing improve, it is increasingly common to perform long-read first de novo genome assemblies followed by polishing with accurate short reads. We briefly introduce FMLRC2, the successor to the original FM-index Long Read Corrector (FMLRC), and illustrate its performance as a fast and accurate de novo assembly polisher for both bacterial and eukaryotic genomes.

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing.

A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

Polypolish: Short-read polishing of long-read bacterial genome assemblies.

Long-read-only bacterial genome assemblies usually contain residual errors, most commonly homopolymer-length errors. Short-read polishing tools can use short reads to fix these errors, but most rely on short-read alignment which is unreliable in repeat regions. Errors in such regions are therefore challenging to fix and often remain after short-read polishing. Here we introduce Polypolish, a new short-read polisher which uses all-per-read alignments to repair errors in repeat sequences that other polishers cannot. Polypolish performed well in benchmarking tests using both simulated and real reads, and it almost never introduced errors during polishing. The best results were achieved by using Polypolish in combination with other short-read polishers.

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae.

Klebsiella pneumoniae has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and drug susceptible community-acquired invasive infections. These pathotypes are generally associated with two distinct subsets of K. pneumoniae lineages or 'clones' that are distinguished by the presence of acquired resistance genes and several key virulence loci. Genomic evolutionary analyses of the most notorious MDR and invasive community-associated ('hypervirulent') clones indicate differences in terms of chromosomal recombination dynamics and capsule polysaccharide diversity, but it remains unclear if these differences represent generalised trends. Here we leverage a collection of >2200 K. pneumoniae genomes to identify 28 common clones (n ≥ 10 genomes each), and perform the first genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified on the basis of acquired resistance and virulence gene prevalence. Chromosomal recombination, surface polysaccharide locus diversity, pan-genome, plasmid and phage dynamics were characterised and compared. The data showed that MDR clones were highly diverse, with frequent chromosomal recombination generating extensive surface polysaccharide locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which also showed a significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. Hence the data indicate that hypervirulent clones may be subject to some sort of constraint for horizontal gene transfer that does not apply to the MDR clones. Our findings are relevant for understanding the risk of emergence of individual K. pneumoniae strains carrying both virulence and acquired resistance genes, which have been increasingly reported and cause highly virulent infections that are extremely difficult to treat. Specifically, our data indicate that MDR clones pose the greatest risk, because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients.

BACKGROUND: Third-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts. METHODS: We performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references. RESULTS: Among 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02). CONCLUSIONS: 3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

GeneMates: an R package for detecting horizontal gene co-transfer between bacteria using gene-gene associations controlled for population structure.

BACKGROUND: Horizontal gene transfer contributes to bacterial evolution through mobilising genes across various taxonomical boundaries. It is frequently mediated by mobile genetic elements (MGEs), which may capture, maintain, and rearrange mobile genes and co-mobilise them between bacteria, causing horizontal gene co-transfer (HGcoT). This physical linkage between mobile genes poses a great threat to public health as it facilitates dissemination and co-selection of clinically important genes amongst bacteria. Although rapid accumulation of bacterial whole-genome sequencing data since the 2000s enables study of HGcoT at the population level, results based on genetic co-occurrence counts and simple association tests are usually confounded by bacterial population structure when sampled bacteria belong to the same species, leading to spurious conclusions. RESULTS: We have developed a network approach to explore WGS data for evidence of intraspecies HGcoT and have implemented it in R package GeneMates ( github.com/wanyuac/GeneMates ). The package takes as input an allelic presence-absence matrix of interested genes and a matrix of core-genome single-nucleotide polymorphisms, performs association tests with linear mixed models controlled for population structure, produces a network of significantly associated alleles, and identifies clusters within the network as plausible co-transferred alleles. GeneMates users may choose to score consistency of allelic physical distances measured in genome assemblies using a novel approach we have developed and overlay scores to the network for further evidence of HGcoT. Validation studies of GeneMates on known acquired antimicrobial resistance genes in Escherichia coli and Salmonella Typhimurium show advantages of our network approach over simple association analysis: (1) distinguishing between allelic co-occurrence driven by HGcoT and that driven by clonal reproduction, (2) evaluating effects of population structure on allelic co-occurrence, and (3) direct links between allele clusters in the network and MGEs when physical distances are incorporated. CONCLUSION: GeneMates offers an effective approach to detection of intraspecies HGcoT using WGS data.

Convergence of virulence and MDR in a single plasmid vector in MDR Klebsiella pneumoniae ST15.

BACKGROUND: MDR and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options. OBJECTIVES: Our aim was to characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15. METHODS: The complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing. RESULTS: Both isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and seven other AMR genes (blaTEM, aac3'-IIa, dfrA1, satA2, blaSHV, sul1 and aadA1). The other carried remnants of these elements encoding blaTEM and aac3'-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania. CONCLUSIONS: The presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance.

Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307.

OBJECTIVES: Recent reports indicate the emergence of a new carbapenemase-producing Klebsiella pneumoniae clone, ST307. We sought to better understand the global epidemiology and evolution of this clone and evaluate its association with antimicrobial resistance (AMR) genes. METHODS: We collated information from the literature and public databases and performed a comparative analysis of 95 ST307 genomes (including 37 that were newly sequenced). RESULTS: We show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the blaCTX-M-15 ESBL gene and several other AMR determinants. CONCLUSIONS: Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance has occasionally emerged.

Deepbinner: Demultiplexing barcoded Oxford Nanopore reads with deep convolutional neural networks.

Multiplexing, the simultaneous sequencing of multiple barcoded DNA samples on a single flow cell, has made Oxford Nanopore sequencing cost-effective for small genomes. However, it depends on the ability to sort the resulting sequencing reads by barcode, and current demultiplexing tools fail to classify many reads. Here we present Deepbinner, a tool for Oxford Nanopore demultiplexing that uses a deep neural network to classify reads based on the raw electrical read signal. This 'signal-space' approach allows for greater accuracy than existing 'base-space' tools (Albacore and Porechop) for which signals must first be converted to DNA base calls, itself a complex problem that can introduce noise into the barcode sequence. To assess Deepbinner and existing tools, we performed multiplex sequencing on 12 amplicons chosen for their distinguishability. This allowed us to establish a ground truth classification for each read based on internal sequence alone. Deepbinner had the lowest rate of unclassified reads (7.8%) and the highest demultiplexing precision (98.5% of classified reads were correctly assigned). It can be used alone (to maximise the number of classified reads) or in conjunction with other demultiplexers (to maximise precision and minimise false positive classifications). We also found cross-sample chimeric reads (0.3%) and evidence of barcode switching (0.3%) in our dataset, which likely arise during library preparation and may be detrimental for quantitative studies that use multiplexing. Deepbinner is open source (GPLv3) and available at https://github.com/rrwick/Deepbinner.

Antimicrobial-Resistant Klebsiella pneumoniae Carriage and Infection in Specialized Geriatric Care Wards Linked to Acquisition in the Referring Hospital.

Background: Klebsiella pneumoniae is a leading cause of extended-spectrum ß-lactamase (ESBL)-producing hospital-associated infections, for which elderly patients are at increased risk. Methods: We conducted a 1-year prospective cohort study, in which a third of patients admitted to 2 geriatric wards in a specialized hospital were recruited and screened for carriage of K. pneumoniae by microbiological culture. Clinical isolates were monitored via the hospital laboratory. Colonizing and clinical isolates were subjected to whole-genome sequencing and antimicrobial susceptibility testing. Results: K. pneumoniae throat carriage prevalence was 4.1%, rectal carriage 10.8%, and ESBL carriage 1.7%, and the incidence of K. pneumoniae infection was 1.2%. The isolates were diverse, and most patients were colonized or infected with a unique phylogenetic lineage, with no evidence of transmission in the wards. ESBL strains carried blaCTX-M-15 and belonged to clones associated with hospital-acquired ESBL infections in other countries (sequence type [ST] 29, ST323, and ST340). One also carried the carbapenemase blaIMP-26. Genomic and epidemiological data provided evidence that ESBL strains were acquired in the referring hospital. Nanopore sequencing also identified strain-to-strain transmission of a blaCTX-M-15 FIBK/FIIK plasmid in the referring hospital. Conclusions: The data suggest the major source of K. pneumoniae was the patient's own gut microbiome, but ESBL strains were acquired in the referring hospital. This highlights the importance of the wider hospital network to understanding K. pneumoniae risk and infection prevention. Rectal screening for ESBL organisms on admission to geriatric wards could help inform patient management and infection control in such facilities.

Kaptive Web: User-Friendly Capsule and Lipopolysaccharide Serotype Prediction for Klebsiella Genomes.

As whole-genome sequencing becomes an established component of the microbiologist's toolbox, it is imperative that researchers, clinical microbiologists, and public health professionals have access to genomic analysis tools for the rapid extraction of epidemiologically and clinically relevant information. For the Gram-negative hospital pathogens such as Klebsiella pneumoniae, initial efforts have focused on the detection and surveillance of antimicrobial resistance genes and clones. However, with the resurgence of interest in alternative infection control strategies targeting Klebsiella surface polysaccharides, the ability to extract information about these antigens is increasingly important. Here we present Kaptive Web, an online tool for the rapid typing of Klebsiella K and O loci, which encode the polysaccharide capsule and lipopolysaccharide O antigen, respectively. Kaptive Web enables users to upload and analyze genome assemblies in a web browser. The results can be downloaded in tabular format or explored in detail via the graphical interface, making it accessible for users at all levels of computational expertise. We demonstrate Kaptive Web's utility by analyzing >500 K. pneumoniae genomes. We identify extensive K and O locus diversity among 201 genomes belonging to the carbapenemase-associated clonal group 258 (25 K and 6 O loci). The characterization of a further 309 genomes indicated that such diversity is common among the multidrug-resistant clones and that these loci represent useful epidemiological markers for strain subtyping. These findings reinforce the need for rapid, reliable, and accessible typing methods such as Kaptive Web. Kaptive Web is available for use at http://kaptive.holtlab.net/, and the source code is available at https://github.com/kelwyres/Kaptive-Web.

Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads.

The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate "hybrid" assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

Gastrointestinal Carriage Is a Major Reservoir of Klebsiella pneumoniae Infection in Intensive Care Patients.

BACKGROUND: Klebsiella pneumoniae is an opportunistic pathogen and leading cause of hospital-associated infections. Intensive care unit (ICU) patients are particularly at risk. Klebsiella pneumoniae is part of the healthy human microbiome, providing a potential reservoir for infection. However, the frequency of gut colonization and its contribution to infections are not well characterized. METHODS: We conducted a 1-year prospective cohort study in which 498 ICU patients were screened for rectal and throat carriage of K. pneumoniae shortly after admission. Klebsiella pneumoniae isolated from screening swabs and clinical diagnostic samples were characterized using whole genome sequencing and combined with epidemiological data to identify likely transmission events. RESULTS: Klebsiella pneumoniae carriage frequencies were estimated at 6% (95% confidence interval [CI], 3%-8%) among ICU patients admitted direct from the community, and 19% (95% CI, 14%-51%) among those with recent healthcare contact. Gut colonization on admission was significantly associated with subsequent infection (infection risk 16% vs 3%, odds ratio [OR] = 6.9, P < .001), and genome data indicated matching carriage and infection isolates in 80% of isolate pairs. Five likely transmission chains were identified, responsible for 12% of K. pneumoniae infections in ICU. In sum, 49% of K. pneumoniae infections were caused by the patients' own unique strain, and 48% of screened patients with infections were positive for prior colonization. CONCLUSIONS: These data confirm K. pneumoniae colonization is a significant risk factor for infection in ICU, and indicate ~50% of K. pneumoniae infections result from patients' own microbiota. Screening for colonization on admission could limit risk of infection in the colonized patient and others.

Inducible colistin resistance via a disrupted plasmid-borne mcr-1 gene in a 2008 Vietnamese Shigella sonnei isolate.

OBJECTIVES: The objective of this study was to assess the presence of mcr-1 in Shigella sonnei isolated in Vietnam. METHODS: WGS data were analysed for the presence of the mcr-1 gene sequence. The association of mcr-1 with a plasmid was assessed by PCR and by conjugation. RESULTS: Through genome sequencing we identified a plasmid-associated inactive form of mcr-1 in a 2008 Vietnamese isolate of Shigella sonnei. The plasmid was conjugated into Escherichia coli and mcr-1 was activated upon exposure to colistin, resulting in highly colistin-resistant transconjugants. CONCLUSIONS: This is the first description of the mcr-1 gene in Shigella, which is atypical given that colistin is not ordinarily used to treat diarrhoea. Our data suggest the mcr-1 gene has been circulating in human-restricted pathogens for some time but likely carries a selective fitness cost.

Bandage: interactive visualization of de novo genome assemblies.

UNLABELLED: Although de novo assembly graphs contain assembled contigs (nodes), the connections between those contigs (edges) are difficult for users to access. Bandage (a Bioinformatics Application for Navigating De novo Assembly Graphs Easily) is a tool for visualizing assembly graphs with connections. Users can zoom in to specific areas of the graph and interact with it by moving nodes, adding labels, changing colors and extracting sequences. BLAST searches can be performed within the Bandage graphical user interface and the hits are displayed as highlights in the graph. By displaying connections between contigs, Bandage presents new possibilities for analyzing de novo assemblies that are not possible through investigation of contigs alone. AVAILABILITY AND IMPLEMENTATION: Source code and binaries are freely available at https://github.com/rrwick/Bandage. Bandage is implemented in C++ and supported on Linux, OS X and Windows. A full feature list and screenshots are available at http://rrwick.github.io/Bandage. CONTACT: rrwick@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data.

BACKGROUND: Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data. RESULTS: ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots. CONCLUSIONS: ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.

How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies.

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing is usually required for perfection. However, the effect of short-read depth on polishing performance is not well understood. Here, we introduce Pypolca (with default and careful parameters) and Polypolish v0.6.0 (with a new careful parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default and Polypolish-careful commonly introduce false-positive errors at low read depth; (2) most of the benefit of short-read polishing occurs by 25× depth; (3) Polypolish-careful almost never introduces false-positive errors at any depth; and (4) Pypolca-careful is the single most effective polisher. Overall, we recommend the following polishing strategies: Polypolish-careful alone when depth is very low (<5×), Polypolish-careful and Pypolca-careful when depth is low (5-25×), and Polypolish-default and Pypolca-careful when depth is sufficient (>25×).

Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies.

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants. They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.

Hybracter: Enabling Scalable, Automated, Complete and Accurate Bacterial Genome Assemblies.

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants (SNVs). They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance (AMR) genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic, and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.