scholarly journals Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples

2019 ◽  
Author(s):  
Kuiama Lewandowski ◽  
Yifei Xu ◽  
Steven T. Pullan ◽  
Sheila F. Lumley ◽  
Dona Foster ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Strong Relationship ◽  
Complete Sequence ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Global Public Health ◽  
Pathogenic Variants ◽  
Oxford Nanopore ◽  
Viral Sequencing

AbstractInfluenza 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 = 4.7×10-5). 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.

scholarly journals Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Kuiama Lewandowski ◽  
Yifei Xu ◽  
Steven T. Pullan ◽  
Sheila F. Lumley ◽  
Dona Foster ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Limit Of Detection ◽  
Clinical Sample ◽  
Strong Relationship ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Global Public Health ◽  
Pathogenic Variants ◽  
Complete Sequences ◽  
Viral Titers

ABSTRACT 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 for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 102 to 103 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (P = 4.7 × 10−5). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [CT] values, <30) and 6/13 samples with low viral titers (CT values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval [CI], 67 to 93%) and 100% specificity (95% CI, 69 to 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 virus titers, we were able to reconstruct >99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.

scholarly journals Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples

2021 ◽  
Author(s):  
Jutte J.C. de Vries ◽  
Julianne R. Brown ◽  
Nicole Fischer ◽  
Igor A. Sidorov ◽  
Sofia Morfopoulou ◽  
...  
Keyword(s):  
De Novo ◽  
Respiratory Infections ◽  
Limit Of Detection ◽  
Bioinformatic Analysis ◽  
Clinical Samples ◽  
Mixed Infections ◽  
Metagenomic Sequencing ◽  
User Friendliness ◽  
Viral Pathogens ◽  
Clinical Diagnostic

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Methods Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analysed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analysed. Results Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. Conclusion A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

scholarly journals Assessment of metagenomic Nanopore and Illumina sequencing for recovering whole genome sequences of chikungunya and dengue viruses directly from clinical samples

2018 ◽  
Vol 23 (50) ◽  
Author(s):  
Liana E. Kafetzopoulou ◽  
Kyriakos Efthymiadis ◽  
Kuiama Lewandowski ◽  
Ant Crook ◽  
Dan Carter ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Clinical Samples ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Front Line ◽  
Genome Sequences ◽  
Dengue Viruses ◽  
Virus Identification ◽  
Reverse Transcription Pcr ◽  
Oxford Nanopore

Background The recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. Aim To investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples. Methods We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study. Results Direct metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. Conclusions This work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.

scholarly journals Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1358
Author(s):  
Leonard Schuele ◽  
Hayley Cassidy ◽  
Erley Lizarazo ◽  
Katrin Strutzberg-Minder ◽  
Sabine Schuetze ◽  
...  
Keyword(s):  
Influenza A ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Genome Coverage ◽  
Sequence Capture ◽  
Shotgun Metagenomic Sequencing ◽  
Oxford Nanopore ◽  
The One ◽  
Targeted Sequence Capture

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

scholarly journals Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples

2018 ◽  
Author(s):  
Liana E. Kafetzopoulou ◽  
Kyriakos Efthymiadis ◽  
Kuiama Lewandowski ◽  
Ant Crook ◽  
Dan Carter ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Clinical Samples ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Front Line ◽  
Genome Sequences ◽  
Oxford Nanopore

AbstractThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION to study the feasibility of whole genome sequencing from clinical samples containing chikungunya or dengue virus, two of the most important arboviruses. Direct metagenomic sequencing of nucleic acid extracts from serum and plasma without viral enrichment allowed for virus and coinfection identification, subtype determination and in the majority of cases elucidated complete or near-complete genomes adequate for phylogenetic analysis. This work demonstrates that metagenomic whole genome sequencing is feasible for over 90% and 80% of chikungunya and dengue virus PCR-positive patient samples respectively. It confirms the feasibility of field metagenomic sequencing for these and likely other RNA viruses, highlighting the applicability of this approach to front-line public health.

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

2020 ◽  
Author(s):  
Nicholas D Sanderson ◽  
Jeremy Swann ◽  
Leanne Barker ◽  
James Kavanagh ◽  
Sarah Hoosdally ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Health Concern ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Entire Genome ◽  
Culture Independent ◽  
Significant Public Health

AbstractThe 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.

scholarly journals Visual and Rapid Diagnosis of Neisseria gonorrhoeae Using Loop-Mediated Isothermal Amplification Combined With a Polymer Nanoparticle–Based Biosensor in Clinical Application

2021 ◽  
Vol 8 ◽  
Author(s):  
Xu Chen ◽  
Qingxue Zhou ◽  
Xueli Wu ◽  
Shuoshi Wang ◽  
Rui Liu ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Point Of Care ◽  
Low Cost ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Global Public Health ◽  
Fixed Temperature ◽  
Polymer Nanoparticle ◽  
Loop Mediated Isothermal Amplification

Neisseriagonorrhoeae is a host-adapted human pathogen that causes sexually transmitted gonorrhea and remains to be a serious global public health challenge, especially in low- and middle-income regions. It is vital to devise a reliable, simple, cost-saving, and easy-to-use assay for detecting the N. gonorrhoeae agent. In the current study, we firstly report a novel approach, loop-mediated isothermal amplification linked with a polymer nanoparticle–based biosensor (LAMP-PNB), that was used for identifying N. gonorrhoeae in clinical samples. The results showed that the LAMP primers based on the orf1 gene were valid for development of the N. gonorrhoeae-LAMP-PNB assay. The detection system with optimal conditions could be performed at a fixed temperature of 64°C for 40 min. The whole process, including genomic DNA preparation (approximately 10 min), LAMP reaction (40 min), and PNB reporting (approximately 2 min), could be accomplished within 60 min. The limit of detection (LoD) of the N. gonorrhoeae-LAMP-PNB assay was 50 copies per test. The specificity of the current assay was 100%, and no cross-reactions to non–N. gonorrhoeae isolates were observed. These results confirmed that the N. gonorrhoeae-LAMP-PNB technique is a reliable, specific, sensitive, rapid, low-cost, and easy-to-use method for detecting gonococci isolates. More importantly, this assay has great potential to develop a point-of-care (POC) testing method in clinical practice, especially in resource-constrained regions.

scholarly journals Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

2021 ◽  
Author(s):  
Leon Peto ◽  
Gillian Rodger ◽  
Daniel P Carter ◽  
Karen L Osman ◽  
Mehmet Yavuz ◽  
...  
Keyword(s):  
High Throughput ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Nanopore Sequencing ◽  
Rt Pcr ◽  
Symptomatic Infection ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

Background LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. Methods We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. Findings The limit of detection of LamPORE was ten genome copies/μl of extracted RNA, which is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9–99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0–100.0%]). Overall, 1.4% (7/514 [0.5–2.9%]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8–98.1%]). Interpretation LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

scholarly journals Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

2020 ◽  
Author(s):  
Leon Peto ◽  
Gillian Rodger ◽  
Daniel P Carter ◽  
Karen L Osman ◽  
Mehmet Yavuz ◽  
...  
Keyword(s):  
High Throughput ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Nanopore Sequencing ◽  
Rt Pcr ◽  
Symptomatic Infection ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/microlitre of extracted RNA. This is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9-99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0-100.0%]). Overall, 1.4% (7/514 [0.5-2.9]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8-98.1]). This indicates that LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

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