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The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org . Each genome was constructed using a novel assembly tool called Viridian ( https://github.com/iqbal-lab-org/viridian ), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

Background: The airway microbiome in severe asthma has not been characterised at species-level by metagenomic sequencing, nor have the relationships between specific species and mucosal immune responses in 'type-2 low', neutrophilic asthma been defined. We performed an integrated species-level metagenomic data with inflammatory mediators to characterise prevalence of dominant potentially pathogenic organisms and host immune responses., Methods: Sputum and nasal lavage samples were analysed using long-read metagenomic sequencing with Nanopore and qPCR in two cross-sectional adult severe asthma cohorts, Wessex (n = 66) and Oxford (n = 30). We integrated species-level data with clinical parameters and 39 selected airway proteins measured by immunoassay and O-link., Results: The sputum microbiome in health and mild asthma displayed comparable microbial diversity. By contrast, 23% (19/81) of severe asthma microbiomes were dominated by a single respiratory pathogen, namely H. influenzae (n = 10), M. catarrhalis (n = 4), S. pneumoniae (n = 4) and P. aeruginosa (n = 1). Neutrophilic asthma was associated with H. influenzae, M. catarrhalis, S. pneumoniae and T. whipplei with elevated type-1 cytokines and proteases; eosinophilic asthma with higher M. catarrhalis, but lower H. influenzae, and S. pneumoniae abundance. H. influenzae load correlated with Eosinophil Cationic Protein, elastase and IL-10. R. mucilaginosa associated positively with IL-6 and negatively with FGF. Bayesian network analysis also revealed close and distinct relationships of H. influenzae and M. catarrhalis with type-1 airway inflammation. The microbiomes and cytokine milieu were distinct between upper and lower airways., Conclusions: This species-level integrated analysis reveals central, but distinct associations between potentially pathogenic bacteria and airways inflammation in severe asthma., (© 2024 The Author(s). Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa and Staphylococcus aureus , and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.

Background: Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders., Methodology/principal Findings: In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles., Conclusion: Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LT was the recipient of a conference travel award from ONT., (Copyright: © 2024 McCabe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Multi-drug-resistant Neisseria gonorrhoeae infection is a significant public health risk. Rapidly detecting N. gonorrhoeae and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of N. gonorrhoeae DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire N. gonorrhoeae genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.

Deserts represent an extreme challenge for photosynthetic life. Despite their aridity, they are often inhabited by diverse microscopic communities of cyanobacteria. These organisms are commonly found in lithic habitats, where they are partially sheltered from extremes of temperature and UV radiation. However, living under the rock surface imposes additional constraints, such as limited light availability, and enrichment of longer wavelengths than are typically usable for oxygenic photosynthesis. Some cyanobacteria from the genus Chroococcidiopsis can use this light to photosynthesize, in a process known as far-red light photoacclimation, or FaRLiP. This genus has commonly been reported from both hot and cold deserts. However, not all Chroococcidiopsis strains carry FaRLiP genes, thus motivating our study into the interplay between FaRLiP and extreme lithic environments. The abundance of sequence data and strains provided the necessary material for an in-depth phylogenetic study, involving spectroscopy, microscopy, and determination of pigment composition, as well as gene and genome analyses. Pigment analyses revealed the presence of red-shifted chlorophylls d and f in all FaRLiP strains tested. In addition, eight genus-level taxa were defined within the encompassing Chroococcidiopsidales, clarifying the phylogeny of this long-standing polyphyletic order. FaRLiP is near universally present in a generalist genus identified in a wide variety of environments, Chroococcidiopsis sensu stricto, while it is rare or absent in closely related, extremophile taxa, including those preferentially inhabiting deserts. This likely reflects the evolutionary process of gene loss in specialist lineages., (© 2023. ISME Publications B.V.)

Complete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) and short-read (Illumina) datasets. Being able to use nanopore-only data would be a significant advance. Oxford Nanopore Technologies (ONT) have recently released a new flowcell (R10.4) and chemistry (Kit12), which reportedly generate per-read accuracies rivalling those of Illumina data. To evaluate this, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa and Staphylococcus aureus , using Illumina and ONT's R9.4.1/Kit10, R10.3/Kit12, R10.4/Kit12 flowcells/chemistries. We compared raw read accuracy and assembly accuracy for each modality, considering the impact of different nanopore basecalling models, commonly used assemblers, sequencing depth, and the use of duplex versus simplex reads. 'Super accuracy' (sup) basecalled R10.4 reads - in particular duplex reads - have high per-read accuracies and could be used to robustly reconstruct bacterial genomes without the use of Illumina data. However, the per-run yield of duplex reads generated in our hands with standard sequencing protocols was low (typically <10 %), with substantial implications for cost and throughput if relying on nanopore data only to enable bacterial genome reconstruction. In addition, recovery of small plasmids with the best-performing long-read assembler (Flye) was inconsistent. R10.4/Kit12 combined with sup basecalling holds promise as a singular sequencing technology in the reconstruction of commonly studied bacterial genomes, but hybrid assembly (Illumina+R9.4.1 hac) currently remains the highest throughput, most robust, and cost-effective approach to fully reconstruct these bacterial genomes.

Previous metagenomic studies in asthma have been limited by inadequate sequencing depth for species-level bacterial identification and by heterogeneity in clinical phenotyping. We hypothesize that chronic bacterial airways infection is a key "treatable trait" whose prevalence, clinical phenotype and reliable biomarkers need definition. In this study, we have applied a method for Oxford Nanopore sequencing for the unbiased metagenomic characterization of severe asthma. We optimized methods to compare performance of Illumina MiSeq, Nanopore sequencing, and RT-qPCR on total sputum DNA extracts against culture/MALDI-TOF for analysis of induced sputum samples from highly phenotyped severe asthma during clinical stability. In participants with severe asthma ( n  = 23) H. influenzae was commonly cultured ( n  = 8) and identified as the dominant bacterial species by metagenomic sequencing using an optimized method for Illumina MiSeq and Oxford Nanopore. Alongside superior operational characteristics, Oxford Nanopore achieved near complete genome coverage of H. influenzae and demonstrated a high level of agreement with Illumina MiSeq data. Clinically significant infection was confirmed with validated H. influenzae plasmid-based quantitative PCR assay. H. influenzae positive patients were found to have sputum neutrophilia and lower FeNO. In conclusion, using an optimized method of direct sequencing of induced sputum samples, H. influenzae was identified as a clinically relevant pathogen in severe asthma and was identified reliably using metagenomic sequencing. Application of these protocols in ongoing analysis of large patient cohorts will allow full characterization of this clinical phenotype. IMPORTANCE The human airways were once thought sterile in health. Now metagenomic techniques suggest bacteria may be present, but their role in asthma is not understood. Traditional culture lacks sensitivity and current sequencing techniques are limited by operational problems and limited ability to identify pathogens at species level. We optimized a new sequencing technique-Oxford Nanopore technologies (ONT)-for use on human sputum samples and compared it with existing methods. We found ONT was effective for rapidly analyzing samples and could identify bacteria at the species level. We used this to show Haemophilus influenzae was a dominant bacterium in the airways in people with severe asthma. The presence of Haemophilus was associated with a "neutrophilic" form of asthma - a subgroup for which we currently lack specific treatments. Therefore, this technique could be used to target chronic antibiotic therapy and in research to characterize the full breadth of bacteria in the airways.

Diagnosis of orthopedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximize the amount of microbial DNA sequence data available for analysis. Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Of 114 samples generating sequence data, species-level positive percent agreement between metagenomic sequencing and culture was 50/65 (77%; 95% confidence interval [CI], 65 to 86%) and negative percent agreement was 103/114 (90%; 95% CI, 83 to 95%). Saponin treatment reduced the proportion of human bases sequenced in comparison to 5-μm filtration from a median (interquartile range [IQR]) of 98.1% (87.0% to 99.9%) to 11.9% (0.4% to 67.0%), improving reference genome coverage at a 10-fold depth from 18.7% (0.30% to 85.7%) to 84.3% (12.9% to 93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.

Metagenomic sequencing is frequently claimed to have the potential to revolutionize microbiology through rapid species identification and antimicrobial resistance (AMR) prediction. We assess the progress toward these developments. We perform a systematic review and meta-analysis of all published literature on culture-independent metagenomic sequencing for pathogen-agnostic infectious disease diagnostics up to 12 August 2020. Methodologic bias and applicability were assessed using the tool Quadas-2. (Prospero CRD42020163777). A total of 2,023 clinical samples from 13/21 eligible diagnostic test accuracy studies were included in the meta-analysis. Reference standards were culture, molecular testing, clinical decision, or a composite measure. Sensitivity and specificity in the most widely investigated sample types were 90% (95% confidence interval [CI], 78% to 96%) and 86% (45% to 98%) for blood, 75% (54% to 89%) and 96% (72% to 100%) for cerebrospinal fluid (CSF), and 84% (79% to 88%) and 67% (38% to 87%) for orthopedic samples, respectively. We identified a limited use of controls, especially negative controls which were used in only 62% (13/21) of studies. AMR prediction and comparison to phenotypic results were undertaken in four studies; categorical agreement was 88%(80% to 97%), and very major and major error rates were 24% (8% to 40%) and 5% (0% to 12%), respectively. Better human DNA depletion methods are required; a median 91% (interquartile range [IQR], 82% to 98%; range, 76% to 98%) of sequences was classified as human. The median (IQR; range) time from sample to result was 29 hours (24 to 94; 4 to 144 hours). The reported consumable cost per sample ranged from $130 to $685. There is scope for improving the quality of reporting in clinical metagenomic studies. Although our results are limited by the heterogeneity displayed, our results reflect a promising outlook for clinical metagenomics. Methodological improvements and convergence around protocols and best practices may improve performance in the future.

BackgroundInfluenza virus presents a considerable challenge to public health by causing seasonal epidemics and occasional pandemics. Nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid infection diagnosis, rationalising antimicrobial therapy, and supporting infection-control interventions.AimTo evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings.MethodsWe conducted Oxford Nanopore Technologies (Oxford, United Kingdom (UK)) metagenomic sequencing for 180 respiratory samples from a UK hospital during the 2018/19 influenza season, and compared results to routine molecular diagnostic standards (Xpert Xpress Flu/RSV assay; BioFire FilmArray Respiratory Panel 2 assay). We investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data.ResultsCompared to standard testing, Nanopore metagenomic sequencing was 83% (75/90) sensitive and 93% (84/90) specific for detecting influenza A viruses. Of 59 samples with haemagglutinin subtype determined, 40 were H1 and 19 H3. We identified an influenza A(H3N2) genome encoding the oseltamivir resistance S331R mutation in neuraminidase, potentially associated with an emerging distinct intra-subtype reassortant. Whole genome phylogeny refuted suspicions of a transmission cluster in a ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant community-circulating strain. We also detected other potentially pathogenic viruses and bacteria from the metagenome.ConclusionNanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. Full genome generation can help investigate and manage nosocomial outbreaks.

Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis -positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 10 5 Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 10 1 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 10 4 BCG cells/ml; >91% coverage (1× depth) occurred at 10 3 BCG cells/ml. Final validation employed M. tuberculosis -positive clinical samples ( n  = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer., (Copyright © 2020 George et al.)

The rise of antimicrobial-resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here, we show an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20× reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection., (© 2020 Sanderson et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Human metapneumovirus (HMPV) infection causes a spectrum of respiratory tract disease, and may be a significant pathogen in the context of immunocompromise. Here, we report direct-from-sample metagenomic sequencing of HMPV using Oxford Nanopore Technology., Methods: We applied this sequencing approach to 25 respiratory samples that had been submitted to a clinical diagnostic laboratory in a UK teaching hospital. These samples represented 13 patients under the care of a haematology unit over a 20-day period in Spring 2019 (two sampled twice), and ten other patients elsewhere in the hospital between 2017-2019., Results: We generated HMPV reads from 20/25 samples (sensitivity 80% compared to routine diagnostic testing) and retrieved complete HMPV genomes from 15/20 of these. Consensus sequences from Nanopore data were identical to those generated by Illumina, and represented HMPV genomes from two distinct sublineages, A2b and B2. Sequences from ten haematology patients formed a unique genetic group in the A2b sublineage, not previously reported in the UK. Among these, eight HMPV genomes formed a cluster (differing by ≤3 SNPs), likely to reflect nosocomial transmission, while two others were more distantly related and may represent independent introductions to the haematology unit., Conclusion: Nanopore metagenomic sequencing can be used to diagnose HMPV infection, although more work is required to optimise sensitivity. Improvements in the use of metagenomic sequencing, particularly for respiratory viruses, could contribute to antimicrobial stewardship. Generation of full genome sequences can be used to support or rule out nosocomial transmission, and contribute to improving infection prevention and control practices., Competing Interests: Declaration of Competing Interest MC, RV and STP have previously received consumables free of charge from ONT, but not for the study presented here., (Copyright © 2020. Published by Elsevier Ltd.)

Cattle possess the most diverse repertoire of NK cell receptor genes among all mammals studied to date. Killer cell receptor genes encoded within the NK complex and killer cell Ig-like receptor genes encoded within the leukocyte receptor complex have both been expanded and diversified. Our previous studies identified two divergent and polymorphic KLRA alleles within the NK complex in the Holstein-Friesian breed of dairy cattle. By examining a much larger cohort and other ruminant species, we demonstrate the emergence and fixation of two KLRA allele lineages ( KLRA*01 and - *02 ) at a single locus during ruminant speciation. Subsequent recombination events between these allele lineages have increased the frequency of KLRA*02 extracellular domains. KLRA*01 and KLRA*02 transcription levels contrasted in response to cytokine stimulation, whereas homozygous animals consistently transcribed higher levels of KLRA , regardless of the allele lineage. KLRA*02 mRNA levels were also generally higher than KLRA*01 Collectively, these data point toward alternative functional roles governed by KLRA genotype and allele lineage. On a background of high genetic diversity of NK cell receptor genes, this KLRA allele fixation points to fundamental and potentially differential function roles., (Copyright © 2020 The Authors.)

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae -spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥10 4 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture., (Copyright © 2020 Street et al.)

The leukocyte receptor complex (LRC) encodes a large number of immunoglobulin (Ig)-like receptors involved in the immune response, particularly in modulating natural killer (NK) cell function. The killer cell Ig-like receptors ( KIR ), the leukocyte Ig-like receptors ( LILR ), and a recently described novel Ig-like receptor family are highly variable between species, which is consistent with rapid evolution driven by selection pressure from pathogens. Among the species studied to date, only simians (such as humans) and bovids (such as cattle and goats) have an expanded complement of KIR genes and represent an interesting model to study KIR evolution. Using recently improved genome assemblies and an assembly of bacterial artificial chromosomes, we describe the structure of the LRC, and the KIR region in particular, in goats and compare this to sheep as the assemblies allow. These species diverged from a common ancestor ~10 million years ago and from cattle ~25 million years ago. We identified conserved KIR genes common to both goats and sheep and confirm a partial sheep haplotype shared between the Rambouillet and Texel breeds. Goats and sheep have independently expanded two novel KIR subgroups, and unlike cattle or any other mammal, they do not appear to possess a functional 3DL-lineage KIR gene. Investigation of LRC gene expression using available transcriptomic data for various sheep and goat tissues largely confirmed putative gene annotation and revealed that a relatively conserved caprinae -specific KIR subgroup is expressed in macrophages. The LILR and novel Ig-like receptors were also highly expressed across a diverse range of tissues. This further step toward our understanding of the LRC receptor repertoire will help inform future studies investigating immune response variation in these species., (Copyright © 2019 Schwartz, Sanderson, Bickhart, Smith and Hammond.)

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family Enterobacteriaceae , as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.

Pyomyositis is a severe bacterial infection of skeletal muscle, commonly affecting children in tropical regions, predominantly caused by Staphylococcus aureus . To understand the contribution of bacterial genomic factors to pyomyositis, we conducted a genome-wide association study of S. aureus cultured from 101 children with pyomyositis and 417 children with asymptomatic nasal carriage attending the Angkor Hospital for Children, Cambodia. We found a strong relationship between bacterial genetic variation and pyomyositis, with estimated heritability 63.8% (95% CI 49.2-78.4%). The presence of the Panton-Valentine leucocidin (PVL) locus increased the odds of pyomyositis 130-fold (p=10 -17.9 ). The signal of association mapped both to the PVL-coding sequence and to the sequence immediately upstream. Together these regions explained over 99.9% of heritability (95% CI 93.5-100%). Our results establish staphylococcal pyomyositis, like tetanus and diphtheria, as critically dependent on a single toxin and demonstrate the potential for association studies to identify specific bacterial genes promoting severe human disease., Competing Interests: BY, SE, SS, PS, VK, SH, VS, RB, NS, LB, NS, KE, CP, EN, PT, RB, DC, DW, ND, DW, CM No competing interests declared, (© 2019, Young et al.)

Background: Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections but potentially useful for any microbial sequencing, and compare detection by direct-from-clinical-sample metagenomic nanopore sequencing with Illumina sequencing and standard microbiological diagnostic techniques., Results: DNA was extracted from the sonication fluids of seven explanted orthopaedic devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (> 90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing., Conclusions: We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data and use of this pipeline to show initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. The high proportion of human DNA in prosthetic joint infection extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling. The nine samples sequenced in this pilot study have shown a proof of concept for sequencing and analysis that will enable us to investigate further sequencing to improve specificity and sensitivity.

The increasing number of Killer Immunoglobulin-like Receptor (KIR) sequences available for non-human primate species and cattle has prompted development of a centralized database, guidelines for a standardized nomenclature, and minimum requirements for database submission. The guidelines and nomenclature are based on those used for human KIR and incorporate modifications made for inclusion of non-human species in the companion IPD-NHKIR database. Included in this first release are the rhesus macaque (Macaca mulatta), chimpanzee (Pan troglodytes), orangutan (Pongo abelii and Pongo pygmaeus), and cattle (Bos taurus).

We describe a gonorrhoea case with combined high-level azithromycin resistance and ceftriaxone resistance. In February 2018, a heterosexual male was diagnosed with gonorrhoea in the United Kingdom following sexual intercourse with a locally resident female in Thailand and failed treatment with ceftriaxone plus doxycycline and subsequently spectinomycin. Resistance arose from two mechanisms combining for the first time in a genetic background similar to a commonly circulating strain. Urgent action is essential to prevent further spread.

Culture of multiple periprosthetic tissue samples is the current gold standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through culture of sonication fluid from explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of total DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 derivation samples, we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and confirmed our findings in 47 independent validation samples. Compared to results from sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69 (88%; 95% confidence interval [CI], 77 to 94%; for derivation samples 35/38 [92%; 95% CI, 79 to 98%]; for validation samples, 26/31 [84%; 95% CI, 66 to 95%]), and genus-level sensitivity was 64/69 (93%; 95% CI, 84 to 98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97 (88%; 95% CI, 79 to 93%; for derivation samples, 43/50 [86%; 95% CI, 73 to 94%]; for validation samples, 42/47 [89%; 95% CI, 77 to 96%]). High levels of human DNA contamination were seen despite the use of laboratory methods to remove it. Rigorous laboratory good practice was required to minimize bacterial DNA contamination. We demonstrate that metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings, combined with the increasing availability of portable, random-access sequencing technology, offer the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI., (Copyright © 2017 Street et al.)

Primate and rodent NK cells form highly heterogeneous lymphocyte populations owing to the differential expression of germline-encoded receptors. Many of these receptors are polymorphic and recognize equally polymorphic determinants of MHC class I. This diversity can lead to individuals carrying NK cells with different specificities. Cattle have an unusually diverse repertoire of NK cell receptor genes predicted to encode receptors that recognize MHC class I. To begin to examine whether this genetic diversity leads to a diverse NK cell population, we isolated peripheral NK cells from cattle with different MHC homozygous genotypes. Cytokine stimulation differentially influenced the transcription of five receptors at the cell population level. Using dilution cultures, we found that a further seven receptors were differentially transcribed, including five predicted to recognize MHC class I. Moreover, there was a statistically significant reduction in killer cell lectin-like receptor mRNA expression between cultures with different CD2 phenotypes and from animals with different MHC class I haplotypes. This finding confirms that cattle NK cells are a heterogeneous population and reveals that the receptors creating this diversity are influenced by the MHC. The importance of this heterogeneity will become clear as we learn more about the role of NK cells in cattle disease resistance and vaccination., (Copyright © 2015 The Authors.)

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig-like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle., (Copyright © 2014 The Authors.)

Background: Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template., Results: The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region., Conclusions: We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Identification of specific and primary chromosomal alterations during the course of neoplastic development is an essential part of defining the genetic basis of cancer. We have developed a transgenic mouse model for liver neoplasia in which chromosomal lesions associated with both the initial stages of the neoplastic process and the acquisition of malignancy can be analyzed. Here we analyze chromosomal alterations in 11 hepatocellular carcinomas from the c-myc/TGF-alpha double-transgenic mice by fluorescent in situ hybridization with whole chromosome probes, single-copy genes, and 4'-6-diamidino-2-phenylindole (DAPI-) and G-banded chromosomes and report nonrandom cytogenetic alterations associated with the tumor development. All tumors were aneuploid and exhibited nonrandom structural and numerical alterations. A balanced translocation t(5:6)(G1;F2) was identified by two-color fluorescent in situ hybridization in all tumors, and, using a genomic probe, the c-myc transgene was localized near the breakpoint on derivative chromosome der 6. Partial or complete loss of chromosome 4 was observed in all tumors with nonrandom breakage in band C2. Deletions of chromosome 1 were observed in 80% of the tumors, with the most frequent deletion at the border of bands C4 and C5. An entire copy of chromosome 7 was lost in 80% of the tumors cells. Eighty-five percent of the tumor cells had lost one copy of chromosome 12, and the most common breakpoint on chromosome 12 occurred at band D3 (28%). A copy of chromosome 14 was lost in 72%, and band 14E1 was deleted in 32% of the tumor cells. The X chromosome was lost in the majority of the tumor cells. The most frequent deletion on the X chromosome involved band F1. We have previously shown that breakages of chromosomes 1, 6, 7, and 12 were observed before the appearance of morphologically distinct neoplastic liver lesions in this transgenic mouse model. Thus breakpoints on chromosome 4, 9, 14, and X appear to be later events in this model of liver neoplasia. This is the first study to demonstrate that specific sites of chromosomal breakage observed during a period of chromosomal instability in early stages of carcinogenesis are later involved in stable rearrangements in solid tumors. The identification of the 5;6 translocation in all of the tumors has a special significance, being the first balanced translocation reported in human and mouse hepatocellular carcinoma and having the breakpoint near a tumor susceptibility gene and myc transgene site of integration. Moreover, its early occurrence indicates that this is a primary and relevant alteration to the initiation of the neoplastic process. In addition, the concordance between the breakpoints observed during the early dysplastic stage of hepatocarcinogenesis and the stable deletions of chromosomes 1, 4, 6, 7, 9, and 12 in the tumors provides evidence for preferential site of genetic changes in hepatocarcinogenesis.

It has been demonstrated that co-expression of c-myc and transforming growth factor alpha (TGF-alpha) as transgenes in the mouse liver results in a tremendous acceleration of neoplastic development in this organ as compared to expression of either transgene alone [Murakami, H., et al. (1993) Cancer Res., 53, 1719-1723]. In order to clarify the roles of transgenes and additional other genetic alterations during hepatocarcinogenesis, we analyzed liver tumors developed in albumin/c-myc transgenic mice and albumin/c-myc and MT-1/TGF-alpha double transgenic mice. High expression of TGF-alpha transgene was found in nine of 14 (64%) liver tumors in double transgenic mice, suggesting that TGF-alpha overexpression confers growth advantage during hepatocarcinogenesis. Only one of 14 (7%) liver tumors in double transgenic mice and none of 13 liver tumors in c-myc transgenic mice showed overexpression of insulin-like growth factor II (IGF-II). This result was in contrast to the report by Takagi et al. [Takagi, H., et al. (1992) Cancer Res., 52, 5171-5177] which showed overexpression of IGF-II in 75% of liver tumors in TGF-alpha transgenic mice and suggested that the presence of c-myc transgenes together with TGF-alpha from an early stage of hepatocarcinogenesis may lead to different carcinogenic pathways which are independent of IGF-II overexpression. Expression of c-myc transgene was found in most of the liver tumors, but at lower levels than non-tumorous parts of the liver in c-myc and double transgenic mice. These results suggest that c-myc transgene expression cooperates with TGF-alpha in the early stages of hepatocarcinogenesis but has growth disadvantage in later stages of hepatocarcinogenesis. There was no evidence of mutational activation of the H-ras gene or mutational inactivation of the p53 gene in any liver tumors developed in c-myc or double transgenic mice.

The cooperation of the c-myc oncogene with the growth factor transforming growth factor (TGF)-alpha in development of liver tumors in transgenic mice has been demonstrated previously. In this study, we analyzed the ploidy and karyotype of c-myc, TGF-alpha, parental control, and the double transgenic c-myc/TGF-alpha hepatocytes at 3 weeks of age when the liver is histologically normal and at 10 weeks when the c-myc/TGF-alpha liver is dysplastic and contains basophilic foci. Eighty % of the 10-week hepatocytes were aneuploid, and 32% had chromosomal breakage. Statistically significant breakage was observed in six different chromosomes. Breakage at band A5 and at the border of bands C4/5 of chromosome 1 was observed. Fragile sets on chromosome 4 were most frequent in the middle of the chromosome at bands C2 and C6. Chromosome 6 was fragile at band F2. The region of chromosome 7 at bands B5 and D3 was frequently broken and involved in translocations. Chromosome 12 was broken at bands D1 and D3. The breakage sites on chromosomes 1, 4, 7, and 12 correspond to sites of tumor susceptibility genes in the mouse. Although there was no consistent change in copy number, recurrent translocations between chromosomes 1, 4, 7, 12 and 19 were also observed. These studies demonstrate that the development of dysplasia and basophilic foci in the liver is correlated with aneuploidy and chromosome breakage. The specific fragile sites indicate genetic regions that are altered during early stages of hepatocarcinogenesis. Due to the conservation of genetic linkage groups between mice and humans, the identification of genetic alterations in the mouse during hepatocarcinogenesis may provide critical information about tumor susceptibility genes that are important in the early development of human hepatocellular carcinoma.

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc complementary DNA and mouse metallothionein 1 promoter-human transforming growth factor alpha complementary DNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and transforming growth factor alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and transforming growth factor alpha, and possibly other combinations of nuclear oncogenes and growth factors, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion.

Derivatives of both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxyphenacetin (N-OH-P) were tested for their ability to cause DNA damage in Reuber (H4-II-E) cells using the alkaline elution technique. Reuber cells are devoid of N-OH-AAF deacylase, N,O-acyltransferase, and sulfotransferase activities. The hydroxamic acids themselves caused very little DNA damage, while N-hydroxy-2-aminofluorine (20 to 100 microM), N-hydroxyphenetidine (20 to 200 microM), and p-nitrosophenetole (10 to 100 microM) all caused dose-dependent damage. The dose-dependent DNA damage caused by N-acetoxy-2-acetylaminofluorene (5 to 25 microM) was completely inhibited by the deacylase inhibitor paraoxon (100 microM). In the presence of both partially purified rabbit liver cytosolic N,O-acyltransferase and guinea pig liver microsomal deacylase, N-OH-AAF was genotoxic. Neither paraoxon nor tRNA had any effect on the DNA damage induced by N-OH-AAF in the presence of N,O-acyltransferase, while paraoxon completely inhibited the damage when N-OH-AAF was incubated in the presence of guinea pig deacylase, and N-OH-P only caused slight DNA damage at higher concentrations of enzyme. In addition, partially purified guinea pig liver deacylase and N-OH-AAF (25 microM) caused 2600 revertants in the Salmonella test system, while only 380 revertants were seen with a 40-fold greater concentration of N-OH-P (1000 microM). The mutagenicity of both N-OH-AAF and N-OH-P was completely inhibited by paraoxon. Thus, it is clear that metabolites of N-OH-AAF formed outside the cell are capable of passing both the cellular and nuclear membranes to cause genotoxicity. Metabolic activation of N-OH-AAF by either the membrane-bound deacylase or the cytosolic N,O-acyltransferase caused genotoxicity via a deacetylation process. Metabolic activation of N-OH-P by guinea pig deacylase caused low levels of DNA damage, whereas activation by N,O-acyltransferase was not sufficient to cause genotoxicity.

The kinetics of 2-acetylaminofluorene (AAF) metabolism have been studied in liver microsomes from four human subjects over a substrate range of 0.02-200 microM. The N-hydroxylation of AAF was best described by a single enzyme system with a mean Km of 1.63 microM and a mean Vmax of 61 pmol mg-1 protein min-1. Biphasic kinetics for the 7-hydroxylation of AAF in all four subjects were observed and a two enzyme system best described the data. The mean Km and Vmax for the high affinity, low-capacity enzyme were 0.69 microM and 30 pmol mg-1 protein min-1, respectively, while for the low affinity, high capacity enzyme the mean Km was 75 microM and the mean Vmax was 286 pmol mg-1 protein min-1. In one of the four subjects studied the 5-hydroxylation of AAF was similarly resolved into a two enzyme system. The 1-hydroxylation of AAF in human microsomes was a major reaction and was best described by a single enzyme system. The 3- and 5-hydroxylations of AAF were minor metabolic pathways. Non-classical Michaelis-Menten kinetics were observed for the 9-hydroxylation of AAF and at high substrate concentrations this was the major metabolite formed. These data demonstrate the involvement of at least two forms of human cytochrome P450 in the metabolism of AAF.