Oxford Nanopore MinION Research Articles

The Salmonella enterica Plasmidome as a Reservoir of Antibiotic Resistance.

The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.

Complete mitochondrial genomes of five subspecies of the Eurasian magpie Pica pica, obtained with Oxford Nanopore MinION, and their interpretation regarding intraspecific taxonomy

The complete mitochondrial (mt) genomes of five subspecies of the Eurasian (Common) magpie Pica pica were determined for the first time. Lengths of the circular genomes comprise 13 protein-coding genes, two rRNA genes (for 12S rRNA and 16S rRNA), 22 tRNA genes, and the non-coding control region (CR). Gene content and lengths of the genomes (16,936–16,945 bp) are similar to typical vertebrate mt genomes. The subspecies studied differs by several single substitutions and indels, especially in the CR. The phylogenetic tree based on complete mt genomes shows a deep divergence of the two groups of subspecies which supports the proposed division into two distinct species: P. pica and P. serica.

Deep splicing plasticity of the human adenovirus type 5 transcriptome as a driver of virus evolution

Viral genomes are characterised by having high gene density and complex transcription strategies. One of the most complex is adenovirus which has a double stranded DNA genome and is the archetypal viral system in which splicing was first discovered. Understanding the transcriptional landscape using conventional mRNA cloning or more recent Illumina-based deep sequencing methods offers insight but also has limitations, including the potential for reverse transcription or PCR amplification artefacts and bias. Here we used direct RNA long read length sequencing on an Oxford Nanopore MinION device to gain a quantitative system-wide overview of transcription and splicing as it dynamically changes during a human adenovirus type 5 infection. This global overview revealed an extensive and hitherto unappreciated complexity of alternative splicing and secondary initiating codon usage. Allied to this, analysis of viral polyadenylation patterns over time showed that most viral transcripts tended to shorter polyadenylation lengths as the infection progressed. Moreover, development and use of an ORF-centric bioinformatics pipeline for analysis of sequenced mRNA, provided both a quantitative and deeper qualitative understanding of the genetic potential of this virus. The data strikingly illustrated that across the viral genome adenovirus made multiple distinctly spliced transcripts that coded for the same ORF. Indeed, as many as 11,000 different splicing patterns were recorded across the viral genome over the three time points analysed. This constitutive low level use of alternative splicing patterns and secondary ORFs potentially enables the virus to maximise its coding potential over evolutionary timescales.

Genome Sequence of SARS-CoV-2 Isolate Cali-01, from Colombia, Obtained Using Oxford Nanopore MinION Sequencing.

We report the genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate obtained from a patient with symptoms of coronavirus disease 2019 (COVID-19) who was infected in Cali, Colombia. The patient had no recent travel record and did not require hospitalization. The virus genome was obtained using Oxford Nanopore MinION sequencing.

Whole Genome Sequencing and Characterization of Multidrug-Resistant (MDR) Bacterial Strains Isolated From a Norwegian University Campus Pond.

The presence of extended-spectrum β-lactamase (ESBL)-producing bacteria in environmental sources has been reported worldwide and constitutes a serious risk of community-acquired infections with limited treatment options. The current study aimed to explore the presence of these worrisome bacteria in a pond located at the Norwegian University of Life Sciences in Ås, Norway. A total of 98 bacterial isolates survived growth on selective chromogenic media and were identified by 16S rRNA Sanger sequencing. All strains were evaluated for the presence of the most commonly found β-lactamases and ESBLs in clinical settings (blaCTX–M groups 1, 2, and 9, blaCMY, blaSHV, and blaTEM) and carbapenemases (blaIMP, blaKPC, blaNDM, blaOXA, blaSFC1, blaVIM) through multiplex PCR. A total of eight strains were determined to contain one or more genes of interest. Phenotypic resistance to 18 antimicrobial agents was assessed and isolates were subjected to whole genome sequencing through a combination of Oxford Nanopore’s MinION and Illumina’s MiSeq. Results revealed the presence of β-lactamase and ESBL-producing Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and a Paraburkholderia spp. Identified β-lactamases and ESBLs include blaCTX–M, blaTEM, blaCMY, blaSHV and a possible blaKPC-like gene, with both documented and novel sequences established. In addition, two inducible β-lactamases were found, a class A β-lactamase (L1) and a cephalosporinase (L2). All strains were determined to be multidrug resistant and numerous resistance genes to non-β-lactams were observed. In conclusion, this study demonstrates that environmental sources are a potential reservoir of clinically relevant ESBL-producing bacteria that may pose a health risk to humans upon exposure.

Complete, high-quality genomes from long-read metagenomic sequencing of two wolf lichen thalli reveals enigmatic genome architecture

Fungal genomes display incredible levels of complexity and diversity, and are exceptional study systems for genome evolution. Here we used the Oxford Nanopore MinION sequencing platform to generate high-quality fungal genomes from complex metagenomic samples of lichen thalli. We sequenced two wolf lichens using one flow cell per sample, generating 17.1 Gbps for Letharia lupina and 14.3 Gbps for Letharia columbiana. The resulting L. lupina genome is one of the most contiguous lichen genomes available to date, with 49.2 Mbp contained on 31 contigs. The L. columbiana genome, while less contiguous, is still relatively high quality, with 52.3 Mbp on a total of 161 contigs. Each thallus for both species contained multiple distinct haplotypes, a phenomenon that has rarely been empirically demonstrated. The Oxford Nanopore sequencing technologies are robust and effective when applied to complex symbioses, and have the potential to fundamentally transform our understanding of fungal genetics.

A Chromosome-Scale Assembly of the Garden Orach (Atriplex hortensis L.) Genome Using Oxford Nanopore Sequencing.

Atriplex hortensis (2n = 2x = 18, 1C genome size ∼1.1 gigabases), also known as garden orach and mountain-spinach, is a highly nutritious, broadleaf annual of the Amaranthaceae-Chenopodiaceae alliance (Chenopodiaceae sensu stricto, subfam. Chenopodioideae) that has spread in cultivation from its native primary domestication area in Eurasia to other temperate and subtropical regions worldwide. Atriplex L. is a highly complex but, as understood now, a monophyletic group of mainly halophytic and/or xerophytic plants, of which A. hortensis has been a vegetable of minor importance in some areas of Eurasia (from Central Asia to the Mediterranean) at least since antiquity. Nonetheless, it is a crop with tremendous nutritional potential due primarily to its exceptional leaf and seed protein quantities (approaching 30%) and quality (high levels of lysine). Although there is some literature describing the taxonomy and production of A. hortensis, there is a general lack of genetic and genomic data that would otherwise help elucidate the genetic variation, phylogenetic positioning, and future potential of the species. Here, we report the assembly of the first high-quality, chromosome-scale reference genome for A. hortensis cv. “Golden.” Long-read data from Oxford Nanopore’s MinION DNA sequencer was assembled with the program Canu and polished with Illumina short reads. Contigs were scaffolded to chromosome scale using chromatin-proximity maps (Hi-C) yielding a final assembly containing 1,325 scaffolds with a N50 of 98.9 Mb – with 94.7% of the assembly represented in the nine largest, chromosome-scale scaffolds. Sixty-six percent of the genome was classified as highly repetitive DNA, with the most common repetitive elements being Gypsy-(32%) and Copia-like (11%) long-terminal repeats. The annotation was completed using MAKER which identified 37,083 gene models and 2,555 tRNA genes. Completeness of the genome, assessed using the Benchmarking Universal Single Copy Orthologs (BUSCO) metric, identified 97.5% of the conserved orthologs as complete, with only 2.2% being duplicated, reflecting the diploid nature of A. hortensis. A resequencing panel of 21 wild, unimproved and cultivated A. hortensis accessions revealed three distinct populations with little variation within subpopulations. These resources provide vital information to better understand A. hortensis and facilitate future study.

Complete Mitochondrial Genome of a Gymnosperm, Sitka Spruce (Picea sitchensis), Indicates a Complex Physical Structure.

Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, with a few notable exceptions. We have sequenced and assembled the complete 5.5-Mb mitochondrial genome of Sitka spruce (Picea sitchensis), to date, one of the largest mitochondrial genomes of a gymnosperm. We sequenced the whole genome using Oxford Nanopore MinION, and then identified contigs of mitochondrial origin assembled from these long reads based on sequence homology to the white spruce mitochondrial genome. The assembly graph shows a multipartite genome structure, composed of one smaller 168-kb circular segment of DNA, and a larger 5.4-Mb single component with a branching structure. The assembly graph gives insight into a putative complex physical genome structure, and its branching points may represent active sites of recombination.

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum \u03b2-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

BackgroundEnvironmental surveillance of antibiotic resistance can contribute towards better understanding and management of human and environmental health. This study applied a combination of long-read Oxford Nanopore MinION and short-read Illumina MiSeq-based sequencing to obtain closed complete genome sequences of two CTX-M-producing multidrug-resistant Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway, in order to understand the potential for mobility of the detected antibiotic resistance genes (ARGs).ResultsThe complete genome sequence of strain 631 (E. coli sequence type 38) was assembled into a circular chromosome of 5.19 Mb and five plasmids (between 98 kb and 5 kb). The majority of ARGs cluster in close proximity to each other on the chromosome within two separate multidrug-resistance determining regions (MDRs), each flanked by IS26 transposases. MDR-1 carries blaTEM-1, tmrB, aac(3)-IId, aadA5, mph(A), mrx, sul1, qacEΔ1 and dfrA17; while MDR-2 harbors aph(3″)-Ib, aph(6)-Id, blaTEM-1, catA1, tet(D) and sul2. Four identical chromosomal copies of blaCTX-M-14 are located outside these regions, flanked by ISEc9 transposases. Strain 1500 (E. coli sequence type 191) exhibited a circular chromosome of 4.73 Mb and two plasmids (91 kb and 4 kb). The 91 kb conjugative plasmid belonging to IncI1 group carries blaCTX-M-15 and blaTEM-1 genes.ConclusionThis study confirms the efficacy of combining Nanopore long-read and Illumina short-read sequencing for determining complete bacterial genome sequences, enabling detection and characterization of clinically important ARGs in the marine environment in Norway, with potential for further dissemination. It also highlights the need for environmental surveillance of antibiotic resistance in low prevalence settings like Norway.

The genome of Mekong tiger perch (Datnioides undecimradiatus) provides insights into the phylogenetic position of Lobotiformes and biological conservation

Mekong tiger perch (Datnioides undecimradiatus) is an ornamental and vulnerable freshwater fish native to the Mekong basin in Indochina, belonging to the order Lobotiformes. Here, we generated 121X stLFR co-barcode clean reads and 18X Oxford Nanopore MinION reads and obtained a 595 Mb Mekong tiger perch genome, which is the first whole genome sequence in the order Lobotiformes. Based on this genome, the phylogenetic tree analysis suggested that Lobotiformes is more closely related to Sciaenidae than to Tetraodontiformes, resolving a long-time dispute. We depicted the genes involved in pigment development in Mekong tiger perch and results confirmed that the four rate-limiting genes of pigment synthesis had been retained after fish-specific genome duplication. We also estimated the demographic history of Mekong tiger perch, which showed that the effective population size suffered a continuous reduction possibly related to the contraction of immune-related genes. Our study provided a reference genome resource for the Lobotiformes, as well as insights into the phylogenetic position of Lobotiformes and biological conservation.

Complete Genome Sequence of Tetracycline-Resistant Serratia liquefaciens S1, Isolated from Mixed Greens, Obtained Using Illumina MiSeq and Oxford Nanopore MinION Sequencing

Serratia liquefaciens strain S1 was isolated from mixed greens and sequenced with short-read Illumina MiSeq technology and long-read MinION technology. Hybrid sequence assembly generated a complete single circular chromosome and two extrachromosomal contigs, which together encoded 5,098 proteins, 85 tRNAs, and 22 rRNAs.

Analysis of the Mouse Y Chromosome by Single-Molecule Sequencing With Y Chromosome Enrichment

Since human and mouse Y chromosomes contain repeated sequences, it is difficult to determine the precise sequences and analyze the function of individual Y chromosome genes. Therefore, the causes of many diseases and abnormalities related to Y chromosome genes, such as male infertility, remain unclear. In this study, to elucidate the mouse Y chromosome, we enriched the mouse Y chromosome using a fluorescence-activated cell sorter (FACS) equipped with commonly used UV and blue 488 nm lasers and read the nucleotides using the Oxford Nanopore MinION long-read sequencer. This sequencing strategy allows us to cover the whole known region as well as the potential undetermined region of the Y chromosome. FACS-based chromosome enrichment and long-read sequencing are suitable for analysis of the Y chromosome sequences and may lead to further understanding of the physiological role of Y chromosome genes.

DeepNano-blitz: a fast base caller for MinION nanopore sequencers.

Oxford Nanopore MinION is a portable DNA sequencer that is marketed as a device that can be deployed anywhere. Current base callers, however, require a powerful GPU to analyze data produced by MinION in real time, which hampers field applications. We have developed a fast base caller DeepNano-blitz that can analyze stream from up to two MinION runs in real time using a common laptop CPU (i7-7700HQ), with no GPU requirements. The base caller settings allow trading accuracy for speed and the results can be used for real time run monitoring (i.e. sample composition, barcode balance, species identification, etc.) or prefiltering of results for more detailed analysis (i.e. filtering out human DNA from human-pathogen runs). DeepNano-blitz has been developed and tested on Linux and Intel processors and is available under MIT license at https://github.com/fmfi-compbio/deepnano-blitz. vladimir.boza@fmph.uniba.sk. Supplementary data are available at Bioinformatics online.

Identification of fosA10, a Novel Plasmid-Mediated Fosfomycin Resistance Gene of Klebsiella pneumoniae Origin, in Escherichia coli.

PurposeSeveral subtypes of plasmid-mediated fosfomycin resistance gene fosA in Enterobacteriaceae have been reported worldwide and have caused concern. The present study characterized a novel member of fosA gene located on a plasmid from Escherichia coli.Materials and MethodsA fosfomycin-resistant E. coli isolate PK9 was recovered from a chicken meat sample in 2018. The presence of fosA genes was detected by PCR and sequencing. Whole-genome sequencing (WGS), conjugation, and cloning were performed to identify the mechanism responsible for fosfomycin resistance. Oxford Nanopore MinION sequencing was carried out to characterize the plasmid carrying fosfomycin resistance gene and the genetic context of the novel fosA variant.ResultsA novel fosA gene with significant homology (>98%) with fosA6 and fosA5 genes was identified by WGS and was named fosA10. FosA10 shared 56.1% to 98.6% amino acid sequence identity with other reported plasmid-mediated FosA enzymes. Fosfomycin resistance and fosA10 gene were successfully transferred to E. coli C600 by conjugation. Cloning confirmed that FosA10 could confer fosfomycin resistance (MIC > 128 μg/mL). The fosA10 gene was localized on a 53kb IncFII (F35:A-:B-) plasmid. The ∆lysR-fosA10-∆hp fragment (4328 bp), located between two copies of IS10R, showed 100% identity with the chromosomal sequences of 17 Klebsiella pneumoniae strains of ST664 and one of ST3821 in GenBank.ConclusionOur findings indicated that the fosA10 gene of E. coli might be captured from the chromosome of K. pneumoniae by IS10, which further demonstrated that K. pneumoniae might act as a reservoir of fosA-like genes acquired by E. coli.

Species-level evaluation of the human respiratory microbiome.

BackgroundChanges to human respiratory tract microbiome may contribute significantly to the progression of respiratory diseases. However, there are few studies examining the relative abundance of microbial communities at the species level along the human respiratory tract.FindingsBronchoalveolar lavage, throat swab, mouth rinse, and nasal swab samples were collected from 5 participants. Bacterial ribosomal operons were sequenced using the Oxford Nanopore MinION to determine the relative abundance of bacterial species in 4 compartments along the respiratory tract. More than 1.8 million raw operon reads were obtained from the participants with ∼600,000 rRNA reads passing quality assurance/quality control (70–95% identify; >1,200 bp alignment) by Discontiguous MegaBLAST against the EZ BioCloud 16S rRNA gene database. Nearly 3,600 bacterial species were detected overall (>750 bacterial species within the 5 dominant phyla: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria. The relative abundance of bacterial species along the respiratory tract indicated that most microbes (95%) were being passively transported from outside into the lung. However, a small percentage (<5%) of bacterial species were at higher abundance within the lavage samples. The most abundant lung-enriched bacterial species were Veillonella dispar and Veillonella atypica while the most abundant mouth-associated bacterial species were Streptococcus infantis and Streptococcus mitis.ConclusionsMost bacteria detected in lower respiratory samples do not seem to colonize the lung. However, >100 bacterial species were found to be enriched in bronchoalveolar lavage samples (compared to mouth/nose) and may play a substantial role in lung health.

Comparison of third-generation sequencing approaches to identify viral pathogens under public health emergency conditions.

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses timely makes it a powerful tool for public health emergency response. Third-generation sequencing (TGS) offers advantages in speed and length of detection over second-generation sequencing (SGS). Here, we presented the end-to-end workflows for both Oxford Nanopore MinION and Pacbio Sequel on a viral disease emergency event, along with Ion Torrent PGM as a reference. A specific pipeline for comparative analysis on viral genomes recovered by each platform was assembled, given the high errors of base-calling for TGS platforms. All the three platforms successfully identified and recovered at least 85% Norovirus GII genomes. Oxford Nanopore MinION spent the least sample-to-answer turnaround time with relatively low but enough accuracy for taxonomy classification. Pacbio Sequel recovered the most accurate viral genome, while spending the longest time. Overall, Nanopore metagenomics can rapidly characterize viruses, and Pacbio Sequel can accurately recover viruses. This study provides a framework for designing the appropriate experiments that are likely to lead to accurate and rapid virus emergency response.

First genomic characterization of a Belgian Enterovirus C104 using sequence-independent Nanopore sequencing

Because of the enormous variation in their genome sequence, genotyping enteroviruses by standard methods can prove to be quite challenging. Nanopore sequencing offers the potential to overcome the limitations of older techniques, but thus far, only amplicon-based strategies have been used to sequence complete enterovirus genomes. By combining a sequence-independent, single primer amplification (SISPA) for cDNA generation with next-generation sequencing using the Oxford Nanopore MinION, complete enterovirus genomes can be obtained in an easy-to-use, sequence-independent manner. To demonstrate its usability, we applied this technique to determine the complete genome sequence of an enterovirus C104 strain, representing the first documented occurrence of this uncommon enterovirus strain in Belgium.

The Genome of Caenorhabditis bovis.

The free-living nematode Caenorhabditis elegans is akey laboratory model for metazoan biology. C.elegans has also become a model for parasitic nematodes despite being only distantly related to most parasitic species. All of the ∼65 Caenorhabditis species currently in culture are free-living, with most having been isolated from decaying plant or fungal matter. Caenorhabditis bovis is a particularly unusual species that has been isolated several times from the inflamed ears of Zebu cattle in Eastern Africa, where it is associated with thedisease bovine parasitic otitis. C.bovis is therefore of particular interest to researchers interested in the evolution of nematode parasitism. However, as C.bovis is not in laboratory culture, it remains little studied. Here, by sampling livestock markets and slaughterhouses in Western Kenya, we successfully reisolated C.bovis from the ear of adult female Zebu. We sequenced the genome of C.bovis using the Oxford Nanopore MinION platform in a nearby field laboratory and used the data to generate a chromosome-scaledraft genome sequence. We exploited this draft genome sequence to reconstruct the phylogenetic relationships of C.bovis to other Caenorhabditis species and reveal the changes in genome size and content that have occurred during its evolution. We also identified expansions in several gene families that have been implicated in parasitism in other nematode species. The high-quality draft genome and our analyses thereof represent a significant advancement in our understanding of this unusual Caenorhabditis species.

Early adaptation strategies of Saccharomyces cerevisiae and Torulaspora delbrueckii to co-inoculation in high sugar grape must-like media

Torulaspora delbrueckii and Saccharomyces cerevisiae are yeast species found concurrently in wine. In order to commence fermentation, they adapt to the initial harsh environment, maintaining cellular homeostasis and promoting metabolism. These actions involve an intricate regulation of stress tolerance, growth and metabolic genes. Their phenotypes are influenced by the fermentation environment and physiological state of the cell, but such gene-environment interactions are poorly understood. This study aimed to compare the cell physiology of the two species, through genome-wide analysis of gene expression, coupling Oxford Nanopore MinION and Illumina Hiseq sequencing platforms. The early transcriptional responses to stress, nutrients and cell-to-cell communication were analysed. Particular attention was given to the fundamental gene modulations, leading to an understanding of the physiological changes needed to maintain cellular homeostasis, exit the quiescent state and establish dominance in the fermentation. Our findings suggest the existence of species-specific adaptation strategies in response to growth in a high sugar synthetic grape juice medium.

Rapid inference of antibiotic resistance and susceptibility by genomic neighbour typing

Surveillance of drug-resistant bacteria is essential for healthcare providers to deliver effective empirical antibiotic therapy. However, traditional molecular epidemiology does not typically occur on a timescale that could affect patient treatment and outcomes. Here, we present a method called ‘genomic neighbour typing’ for inferring the phenotype of a bacterial sample by identifying its closest relatives in a database of genomes with metadata. We show that this technique can infer antibiotic susceptibility and resistance for both Streptococcus pneumoniae and Neisseria gonorrhoeae. We implemented this with rapid k-mer matching, which, when used on Oxford Nanopore MinION data, can run in real time. This resulted in the determination of resistance within 10 min (91% sensitivity and 100% specificity for S. pneumoniae and 81% sensitivity and 100% specificity for N. gonorrhoeae from isolates with a representative database) of starting sequencing, and within 4 h of sample collection (75% sensitivity and 100% specificity for S. pneumoniae) for clinical metagenomic sputum samples. This flexible approach has wide application for pathogen surveillance and may be used to greatly accelerate appropriate empirical antibiotic treatment.