Real-time analysis of nanopore-based metagenomic sequencing from infected orthopaedic devices

Sanderson, Nicholas D.; Street, Teresa; Foster, Dona; Swann, Jeremy; Atkins, Bridget L.; Brent, Andrew; McNally, Martin; Oakley, Sarah; Taylor, Adrian; Peto, Tim E. A.; Crook, Derrick W.; Eyre, David W

doi:10.1186/s12864-018-5094-y

Cited by 119 publications

(98 citation statements)

References 33 publications

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“…Besides its small size and low cost, the intriguing feature of MinION sequencer is that it can provide a real‐time and on‐site analysis of any genetic material, which should be useful especially for clinical applications . With the ability to generate longer read lengths, MinION™ analysis targets the whole coding region of the 16S rRNA gene, showing great potential for rapid pathogen detection with more accuracy and sensitivity . We have previously established a sequencing method and bioinformatics pipeline for rapid determination of bacterial composition based on 16S rRNA gene amplicon sequencing via the MinION™ platform .…”

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confidence: 99%

Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION™ nanopore sequencer

et al. 2019

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Rapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION™ sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable time frame for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate‐limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinION™ sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.

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confidence: 99%

Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION™ nanopore sequencer

et al. 2019

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“…In contrast, single-molecule sequencing platforms such as the Oxford Nanopore MinION, GridION and PromethION are able to sequence very long fragments of DNA (>10 Kbp, with over 2 Mbp reported) [4,5] and with recent improvements to the platform making metagenomic studies using nanopore more viable, such studies are increasing in frequency [6,7,8,9]. Long reads help with alignment-based assignment of taxonomy and function due to their increased information content [10,11].…”

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confidence: 99%

Ultra-deep, long-read nanopore sequencing of mock microbial community standards

Nicholls

Quick

Tang³

et al. 2018

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Background: Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition. Methods: We sequenced two commercially-available mock communities containing ten microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Isolates from the same mock community were sequenced individually with Illumina HiSeq. Data: We generated 14 and 16 Gbp from GridION owcells and 146 and 148 Gbp from PromethION owcells for the even and odd communities respectively. Read length N50 was 5.3 Kbp and 5.2 Kbp for the even and log community, respectively. Basecalls and corresponding signal data are made available (4.2 TB in total). Results: Alignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning. Conclusions: We present ultra-deep, long-read nanopore datasets from a well-de ned mock community. These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines.Whole-genome sequencing of microbial communities (metagenomics) has revolutionised our view of microbial evolution and diversity, with numerous potential applications for microbial ecology, clinical microbiology and industrial biotechnology [1,2]. Typically, metagenomic studies use high-throughput sequencing platforms (e.g. Illumina) [3] which generate very high yield, but of limited read length (100-300 bp).

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“…Classified reads were aligned to reference genomes using Minimap2 19 and filtered to a mapQ of 50 by samtools 20 as described in the CRuMPIT workflow 21 (https://gitlab.com/ModernisingMedicalMicrobiology/CRuMPIT). CRuMPIT was used to generate per species metrics for read numbers, bases, and coverage depth and breadth.…”

Section: Methodsmentioning

confidence: 99%

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

Street

Barker

Sanderson

et al. 2019

Preprint

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20 2 Oxfordshire Sexual Health Service, Oxford University Hospitals NHS Foundation Trust, 21 Oxford, UK g 22 Brighton and Hove sexual health and contraception service, Royal Sussex County Hospital, 23 Brighton, UK h 24 25 26 Running title: Nanopore sequencing N. gonorrhoeae direct from urine 27 Address correspondence to teresa.street@ndm.ox.ac.uk and david.eyre@bdi.ox.ac.uk. 28 3 Abstract 29Background. Empirical gonorrhoea treatment at initial diagnosis reduces onward 30 transmission. However, increasing resistance to multiple antibiotics may necessitate waiting 31 for culture-based diagnostics to select an effective treatment. There is a need for same-day 32 culture-free diagnostics that identify infection and detect antimicrobial resistance. 33 Methods. We investigated if Nanopore sequencing can detect sufficient N. gonorrhoeae 34 DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae 35 spiked urine samples and samples from gonorrhoea infections to determine optimal DNA 36 extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced whilst 37 minimizing contaminating host DNA. 38 Results. In simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio 39 N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA 40 with saponin increased N. gonorrhoeae yields in simulated infections, but decreased yields 41 in clinical samples. In ten urine samples from men with symptomatic urethral gonorrhoea, 42 ≥87% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with 43 ≥92% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections if ≥10 4 44 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was 45 frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR Media 46 tubes and from urethral swabs, and in the presence of simulated Chlamydia co-infection. 47 Conclusion. Using Nanopore sequencing of urine samples from men with urethral 48 gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of 49 samples without the need for culture. 50 51 4 52

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Real-time analysis of nanopore-based metagenomic sequencing from infected orthopaedic devices

Cited by 119 publications

References 33 publications

Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION™ nanopore sequencer

Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION™ nanopore sequencer

Ultra-deep, long-read nanopore sequencing of mock microbial community standards

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

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