Biology subjects

Sanderson, N. D.

Publications and source records attributed to Sanderson, N. D..

14 recordsLinked to original sources

bioRxiv2026

OxBreaker: species-agnostic pipeline for the analysis of outbreaks using nanopore sequencing

Real-time genomic surveillance may mitigate the spread of health-care-associated infections, but whole-genome sequencing costs and the need for specialised expertise constrain its wide implementation in public health. Here we present OxBreaker, an automated and species-agnostic pipeline optimised for the high-resolution analysis of bacterial and plasmid genomes sequenced via Oxford Nanopore Technologies (ONT). OxBreaker streamlines the transition from raw reads to phylogenetic inference through automated reference selection and high-accuracy variant calling. It is accessible via a graphical user interface (GUI) that can be easily installed locally and operated by non-specialists. Benchmarking against technical and biological replicates of high-priority pathogens demonstrates high accuracy, with false positive variant rates reduced to 0-4 single-nucleotide polymorphisms (SNPs) for common species. We further validated the pipeline by accurately characterising previously published clonal and plasmid-mediated outbreaks, reproducing established phylogenies with improved accessibility. By providing a stable, scalable, open-source offline-compatible solution that matches the resolution of short-read platforms while maintaining the speed of long-read technology, OxBreaker is designed to facilitate the adoption of local, real-time genomic surveillance for frontline infection prevention and control.

genomics↗

bioRxiv2024

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing

2.Whole genome reconstruction of bacterial pathogens has become an import tool for tracking antimicrobial resistance spread, however accurate and complete assemblies have only been achievable using hybrid long and short-read sequencing. We have previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcells produced improved assemblies over the R9.4.1/kit10, however they contained too many errors compared to hybrid Illumina-ONT assemblies. ONT have since released the R10.4.1/kit12 flowcells that promises greater accuracy and yield. They have also released newly trained basecallers using native bacterial DNA containing methylation sites intended to fix systematic errors, specifically Adenosine (A) to Guanine (G) and Cytosine (C) to Thymine (T) substitutions. ONT have recommended the use of Bovine Serum Albumin (BSA) during library preparation to improve sequencing yield and accuracy. To evaluate these improvements, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, as well as 12 disparate E. coli clinical samples from different phylogroups and sequence types. These were all sequenced with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. Here we have found the nanopore long read-only R10.4.1 (kit14) assemblies with basecallers trained using native bacterial methylated DNA produce accurate assemblies from 40x depth or higher, sufficient to be cost-effective compared to hybrid long-read (ONT) and short-read (Illumina) sequencing. 3. Impact statementCurrently, the best method of building accurate and complete bacterial genome assemblies is to create a hybrid assembly; combining both long and short DNA sequencing reads. Short reads are more accurate, but can be difficult to assemble into a complete genome. Long reads are generally less accurate, but easier to reconstruct into a complete genome. By combining long and short reads, we get both accuracy and reconstructive power. However, this also involves higher costs and more labour than using a single sequencing platform. In this study, we compare long read only assemblies from Oxford Nanopore Technologys newest iteration of improvements in both chemistry and software to hybrid Illumina-Nanopore assemblies. We sequenced four bacterial pathogens with published reference genomes (Staphylococcus aureus, Klebsiella Pneumoniae, Pseudomonas Aeruginosa, and Escherichia Coli) and twelve bloodstream associated E. coli, and show that assemblies from the newest technology are not only an improvement on the previous iteration, but are able to compete with hybrid Illumina-Nanopore assemblies in their quality, providing a step towards bacterial genome assembly using a single sequencing platform. 4. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article, through supplementary data files, or in publicly accessible repositories. Nanopore and Illumina fastq data are available in the ENA under project accession: PRJEB51164. Assemblies have been made available at: https://figshare.com/articles/dataset/R10_4_1_KIT14_comparison_assemblies/2497 2954 Code and analysis outputs are available at: https://gitlab.com/ModernisingMedicalMicrobiology/assembly_comparison

microbiology↗

bioRxiv2022

Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction

2.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 utilise 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 ONTs 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. 3. Impact statementOur understanding of microbes has been greatly enhanced by the capacity to evaluate their genetic make-up using a technology known as whole genome sequencing. Sequencers represent microbial genomes as stretches of shorter sequence known as reads, which are then assembled using computational algorithms. Different types of sequencing approach have advantages and disadvantages with respect to the accuracy and length of the reads they generate; this in turn affects how reliably genomes can be assembled. Currently, to completely reconstruct bacterial genomes in a high-throughput and cost-effective manner, researchers tend to use two different types of sequencing data, namely Illumina (short-read) and nanopore (long-read) data. Illumina data are highly accurate; nanopore data are much longer, and this combination facilitates accurate and complete bacterial genomes in a so-called "hybrid assembly". However, new developments in nanopore sequencing have reportedly greatly improved the accuracy of nanopore data, hinting at the possibility of requiring only a single sequencing approach for bacterial genomics. Here we evaluate these improvements in nanopore sequencing in the reconstruction of four bacterial reference strains, where the true sequence is already known. We show that although these improvements are extremely promising, for high-throughput, low-cost complete reconstruction of bacterial genomes hybrid assembly currently remains the optimal approach. 4. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article, through supplementary data files, or in publicly accessible repositories. Nanopore fast5 and fastq data are available in the ENA under project accession: PRJEB51164. Assemblies have been made available at: https://figshare.com/articles/online_resource/q20_comparison_genome_assemblies/196838 67. Code and analysis outputs are available at: https://gitlab.com/ModernisingMedicalMicrobiology/assembly_comparison_analysis/-/tree/main (tagged version v0.5.5).

genomics↗

bioRxiv2020

DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

Mycobacterium tuberculosis (MTB) 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 Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99{degrees}C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 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 each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes [≥]1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

microbiology↗

bioRxiv2020

Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies

Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically important genes (e.g. those involved in AMR/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data has suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore (Oxford Nanopore Technologies [ONT]) flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 hours of sequencing, most chromosomes and plasmids had circularised and there was no benefit associated with longer running times. Quality was similar at 12 hours suggesting shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilising shorter run-times in combination with between-library nuclease washes allows at least 36 Enterobacteriaceae isolates to be sequenced per flowcell, significantly reducing the per isolate sequencing cost. Ultimately this will facilitate large-scale studies utilising hybrid assembly advancing our understanding of the genomics of key human pathogens. Data SummaryO_LIRaw sequencing data is available via NCBI under project accession number PRJNA604975. Sample accession numbers are provided in table S1. C_LIO_LIAssemblies are available via Figshare https://doi.org/10.6084/m9.figshare.11816532.v1. C_LI Impact StatementMost existing sequencing data has been acquired from short-read platforms (eg. Illumina). For some species of bacteria, clinically important genes, such as those involved in antibiotic resistance and/or virulence, are carried on plasmids. Whilst Illumina sequencing is highly accurate, it is generally unable to resolve complete genomic structures due to repetitive regions. Hybrid assembly uses long reads to scaffold together short-read contigs, maximising the benefits of both technologies. A major limiting factor to using hybrid assemblies at scale is the cost of sequencing the same isolate with two different technologies. Here we show that high-quality hybrid assemblies can be created for most isolates using significantly shorter run-times than are currently standard. We demonstrate that a simple washing step allows several libraries to be run on the same flowcell, facilitating the ability to take advantage of shorter running times. Adding nuclease means that contamination between libraries is minimal and has no significant effect on the quality of subsequent hybrid assemblies. This approach reduces the cost of acquiring long reads by >30%, paving the way for large-scale studies utilising hybrid assemblies which will likely significantly enhance our understanding of the genomics of important human pathogens.

bioinformatics↗

bioRxiv2020

High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing

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 gonorrhoea 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 demonstrate 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 20x 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.

microbiology↗

bioRxiv2019

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. 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 desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.

molecular biology↗

bioRxiv2019

Optimizing DNA extraction methods for Nanopore sequencing of Neisseria gonorrhoeae direct from urine samples

BackgroundEmpirical gonorrhoea 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.\n\nMethodsWe investigated if Nanopore sequencing can detect sufficient N. gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae spiked urine samples and samples from gonorrhoea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced whilst minimizing contaminating host DNA.\n\nResultsIn simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio 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 ten urine samples from men with symptomatic urethral gonorrhoea, [≥]87% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with [≥]92% coverage breath at [≥]10-fold depth in 7 (70%) samples. In simulated infections if [≥]104 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 Media tubes and from urethral swabs, and in the presence of simulated Chlamydia co-infection.\n\nConclusionUsing Nanopore sequencing of urine samples from men with urethral gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

microbiology↗

bioRxiv2019

Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential of a diagnostic test for influenza which also provides insights on transmission, evolution and drug resistance, and simultaneously detects other viruses. We therefore set out to apply Oxford Nanopore Technology to metagenomic sequencing of respiratory samples. We generated influenza reads down to a limit of detection of 102-103 genome copies/ml in pooled samples, observing a strong relationship between the viral titre and the proportion of influenza reads (p = 4.7x10-5). Applying our methods to clinical throat swabs, we generated influenza reads for 27/27 samples with high-to-mid viral titres (Cycle threshold (Ct) values <30) and 6/13 samples with low viral titres (Ct values 30-40). No false positive reads were generated from 10 influenza-negative samples. Thus Nanopore sequencing operated with 83% sensitivity (95% CI 67-93%) and 100% specificity (95% CI 69-100%) compared to the current diagnostic standard. Coverage of full length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza titres, we were able to reconstruct >99% complete sequence for all eight gene segments. We also detected Human Coronavirus and generated a near complete Human Metapneumovirus genome from clinical samples. While further optimisation is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza and other respiratory viruses.

microbiology↗

bioRxiv2018

Nanopore metagenomic sequencing of full length human metapneumovirus (HMPV) within a unique sub-lineage

Human metapneumovirus (HMPV) has been recognized as an important pathogen which can cause a spectrum of respiratory tract disease. Here, we report Nanopore metagenomic sequencing of the first full length HMPV genome directly from a throat swab from a UK patient with complex lung disease and immunocompromise. We found a predominance (26.4%) of HMPV reads in the metagenomic sequencing data and consequently assembled the full genome at a high depth of coverage (mean 4,786). Through phylogenetic analyses, we identified this HMPV strain to originate from a unique genetic group in A2b, showing the presence of this group in the UK. Our study demonstrated the effectiveness of Nanopore metagenomic sequencing for diagnosing infectious diseases and recovering complete sequences for genomic characterization, highlighting the applicability of Nanopore sequencing in clinical settings.\n\nImportanceNanopore metagenomic sequencing has the potential to evolve as a point-of-care test for a range of infectious diseases. Here, we report the first full length human metapneumovirus (HMPV) genome in the UK sequenced by Nanopore from a non-invasive sample from an immunocompromised patient. We demonstrate the presence of HMPV from a unique genetic group not previously reported from the UK. Our study demonstrates the effectiveness of Nanopore sequencing for diagnosing an infection that was not detected by routine first-line tests in the clinical microbiology laboratory. We report sufficient genomic data to provide insight into the epidemiology of infection and with the potential to inform treatment decisions.

microbiology↗

bioRxiv2018

Panton-Valentine leukocidin is the key determinant of Staphylococcus aureus pyomyositis in a bacterial genome-wide association study

Pyomyositis is a severe bacterial infection of skeletal muscle, commonly affecting children in tropical regions and 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 in 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 the sequence immediately upstream. Together these regions explained > 99.9% of heritability. Our results establish staphylococcal pyomyositis, like tetanus and diphtheria, as critically dependent on expression of a single toxin and demonstrate the potential for association studies to identify specific bacterial genes promoting severe human disease.

microbiology↗

bioRxiv2018

Control of artefactual variation in reported inter-sample relatedness during clinical use of a Mycobacterium tuberculosis sequencing pipeline

Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artefactual variation between M. tuberculosis isolates during routine next generation sequencing of Mycobacterium spp, we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted, sequenced, reads mapped, and consensus sequences determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of non-Mycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of non-Mycobacterial bacterial DNA, we found significant increases in minor variant frequencies of more than 1.5 fold in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high variation regions strongly influenced by the amount of non-Mycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we have demonstrated an approach identifying critical genomic regions contributing to clinically relevant artefactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multi-step laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

microbiology↗

bioRxiv2017

Real-time analysis of nanopore-based metagenomic sequencing from orthopaedic device infection

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 compare direct-from-clinical-sample metagenomic Illumina sequencing with Nanopore sequencing, and report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections.\n\nDNA 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.\n\nThe 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.\n\nWe demonstrate initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data. The high proportion of human DNA in extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling.

microbiology↗

bioRxiv2017

Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing

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 sonication fluid culture of 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 complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI.\n\nWe compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopaedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent samples.\n\nCompared to sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69(88%,95%CI 77-94%) (derivation samples 35/38[92%,79-98%]; validation 26/31[84%,66-95%]), and genus-level sensitivity was 64/69(93%,84-98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97(88%,79-93%) (derivation 43/50[86%,73-94%], validation 42/47[89%,77-96%]). High levels of human DNA contamination were seen despite use of laboratory methods to remove it. Rigorous laboratory good practice was required to prevent bacterial DNA contamination.\n\nWe demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

microbiology↗