Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular method for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, ‘SMART-9N’, and a version compatible with barcoded PCR primers available from Oxford Nanopore Technologies, ‘Rapid SMART-9N’, for the detection, characterization, and whole-genome sequencing of RNA viruses. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6e00 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

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Coxsackievirus A16 in Southern Vietnam

Frontiers in Microbiology ◽

10.3389/fmicb.2021.689658 ◽

2021 ◽

Vol 12 ◽

Author(s):

Le Nguyen Truc Nhu ◽

Le Nguyen Thanh Nhan ◽

Nguyen To Anh ◽

Nguyen Thi Thu Hong ◽

Hoang Minh Tu Van ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Vaccine Development ◽

Asia Pacific ◽

Health Concern ◽

Clinical Samples ◽

Whole Genome ◽

Evolutionary Analysis ◽

Coxsackievirus A16 ◽

Outbreak Response

Background: Hand, Foot and Mouth Disease (HFMD) is a major public health concern in the Asia-Pacific region. Most recent HFMD outbreaks have been caused by enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16), CVA10, and CVA6. There has been no report regarding the epidemiology and genetic diversity of CVA16 in Vietnam. Such knowledge is critical to inform the development of intervention strategies.Materials and Methods: From 2011 to 2017, clinical samples were collected from in- and outpatients enrolled in a HFMD research program conducted at three referral hospitals in Ho Chi Minh City (HCMC), Vietnam. Throat or rectal swabs positive for CVA16 with sufficient viral load were selected for whole genome sequencing and evolutionary analysis.Results: Throughout the study period, 320 CVA16 positive samples were collected from 2808 HFMD patients (11.4%). 59.4% of patients were male. The median age was 20.8 months (IQR, 14.96–31.41). Patients resided in HCMC (55.3%), Mekong Delta (22.2%), and South East Vietnam (22.5%). 10% of CVA16 infected patients had moderately severe or severe HFMD. CVA16 positive samples from 153 patients were selected for whole genome sequencing, and 66 complete genomes were obtained. Phylogenetic analysis demonstrated that Vietnamese CVA16 strains belong to a single genogroup B1a that clusters together with isolates from China, Japan, Thailand, Malaysia, France and Australia. The CVA16 strains of the present study were circulating in Vietnam some 4 years prior to its detection in HFMD cases.Conclusion: We report for the first time on the molecular epidemiology of CVA16 in Vietnam. Unlike EV-A71, which showed frequent replacement between subgenogroups B5 and C4 every 2–3 years in Vietnam, CVA16 displays a less pronounced genetic alternation with only subgenogroup B1a circulating in Vietnam since 2011. Our collective findings emphasize the importance of active surveillance for viral circulation in HFMD endemic countries, critical to informing outbreak response and vaccine development.

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A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture

Viruses ◽

10.3390/v12080895 ◽

2020 ◽

Vol 12 (8) ◽

pp. 895 ◽

Cited By ~ 2

Author(s):

Jalees A. Nasir ◽

Robert A. Kozak ◽

Patryk Aftanas ◽

Amogelang R. Raphenya ◽

Kendrick M. Smith ◽

...

Keyword(s):

Genome Sequencing ◽

Clinical Diagnostics ◽

Viral Rna ◽

Clinical Samples ◽

Genome Coverage ◽

Advantages And Disadvantages ◽

Low Concentrations ◽

Lower Depth ◽

Viable Method ◽

Rna Concentration

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.

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

10.1101/2020.02.07.939322 ◽

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.

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New approach for genomic characterisation of equine sarcoid-derived BPV-1/-2 using nanopore-based sequencing

Virology Journal ◽

10.1186/s12985-021-01735-5 ◽

2022 ◽

Vol 19 (1) ◽

Author(s):

Lien Gysens ◽

Bert Vanmechelen ◽

Maarten Haspeslagh ◽

Piet Maes ◽

Ann Martens

Keyword(s):

Phylogenetic Trees ◽

Wide Spectrum ◽

Clinical Sample ◽

Clinical Samples ◽

Bovine Papillomavirus ◽

Nanopore Sequencing ◽

Veterinary Research ◽

Equine Sarcoid ◽

Potential Association ◽

The One

Abstract Background Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. Methods In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. Results We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. Conclusions By generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.

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DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00670-20 ◽

2020 ◽

Vol 58 (10) ◽

Author(s):

Sophie George ◽

Yifei Xu ◽

Gillian Rodger ◽

Marcus Morgan ◽

Nicholas D. Sanderson ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Low Cost ◽

Clinical Samples ◽

Heat Inactivation ◽

Whole Genome ◽

Genome Coverage ◽

Content Type ◽

C 30 ◽

Validation Experiments

ABSTRACT 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 105 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 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 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.

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

10.1101/2020.04.05.026864 ◽

2020 ◽

Author(s):

Sophie George ◽

Yifei Xu ◽

Gillian Rodger ◽

Marcus Morgan ◽

Nicholas D. Sanderson ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Low Cost ◽

Clinical Samples ◽

Heat Inactivation ◽

Whole Genome ◽

Genome Coverage ◽

Human Dna ◽

Oxford Nanopore ◽

Oxford Nanopore Technologies

ABSTRACTMycobacterium 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°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.

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Two-Pool Multiplex Long-Amplicon Amplification and Full-Genome Sequencing of SARS-CoV-2

10.21203/rs.3.rs-103455/v1 ◽

2020 ◽

Author(s):

Hong-jie Liu ◽

Jin-Hui Li ◽

Yan-Feng Lin ◽

Xiao-Chen Bo ◽

Hong-Bin Song ◽

...

Keyword(s):

Multiplex Pcr ◽

Genome Sequencing ◽

Massively Parallel Sequencing ◽

Clinical Samples ◽

Nanopore Sequencing ◽

Full Genome ◽

Full Genome Sequencing ◽

Genome Sequences ◽

Genomic Variations ◽

Pcr Targeting

Abstract BackgroundSince the first public genome of SARS-CoV-2, over 170,000 genome sequences of the virus have been shared by researchers world-wide (till November 1st 2020). Multiplex PCR targeting SARS-CoV-2 followed by massively parallel sequencing (MPS) and/or nanopore sequencing is a widely used strategy to recover the genome from primary samples. However, the bias of amplification among different amplicons should not be ignored, which might lead to uneven sequencing coverage on the viral genome.MethodsWe aim to develop a novel multiplex PCR panel to achieve an improved coverage evenness of SARS-CoV-2. We adapt long amplicons (~1000-bp) for the panel and thus reduced the number of primer pairs. The panel was validated with clinical samples and sequenced via MPS sequencing systems and a portable nanopore sequencing device MinION. We evaluated the full-genome coverage evenness and its dependence on viral loads of the long amplicon panel; we then compared it with a 98-plex panel provided by the ARTIC network. The accuracy to identify viral genomic variations based on the panel and sequencing with MinION was assessed.ResultsWe developed a two-pool 36-plex panel for full-genome sequencing of SARS-CoV-2, whose amplicon size ranged from 880 to 1027 bp. For samples with a <30 Ct value, >90% viral genome could be recovered with a high sequencing depth (>0.2 mean depth) by using the long-amplicon panel (n = 36), compared with 79-88% highly covered genome region for the ARTIC panel (n = 5). The coverage evenness of the long-amplicon panel was also less affected by low viral titers and not dependent on sequencing data amount. With MinION sequencing, the consensus viral genomes could be reliably recovered. However, a high false positive rate was observed to identify sub-clonal genomic variations with a <0.6 frequency.ConclusionA novel multiplex PCR panel for full-genome sequencing of SARS-CoV-2 with improved coverage evenness and low requirement of data throughput was validated with clinical samples. Amplification of SARS-CoV-2 with the panel followed by MinION sequencing could generate reliable consensus genome sequences, but the detection of non-dominating viral populations within host is error-prone.

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Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

10.1101/2020.02.21.959817 ◽

2020 ◽

Cited By ~ 3

Author(s):

Tran Thi Nhu Thao ◽

Fabien Labroussaa ◽

Nadine Ebert ◽

Philip V’kovski ◽

Hanspeter Stalder ◽

...

Keyword(s):

Reverse Genetics ◽

Yeast Artificial Chromosome ◽

Vaccine Development ◽

Rna Viruses ◽

Rna Virus ◽

Functional Characterization ◽

Clinical Samples ◽

Emerging Viruses ◽

Synthetic Dna ◽

Synthetic Genomics

AbstractReverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability1-3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.

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Viral Metagenomics on Cerebrospinal Fluid

Genes ◽

10.3390/genes10050332 ◽

2019 ◽

Vol 10 (5) ◽

pp. 332 ◽

Cited By ~ 11

Author(s):

Edridge ◽

Deijs ◽

van Zeggeren ◽

Kinsella ◽

Jebbink ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Viral Load ◽

Rna Viruses ◽

Rna Virus ◽

Viral Metagenomics ◽

Clinical Samples ◽

Cns Infections ◽

Dnase Treatment ◽

Low Load ◽

Multiple Samples

Identifying the causative pathogen in central nervous system (CNS) infections is crucial for patient management and prognosis. Many viruses can cause CNS infections, yet screening for each individually is costly and time-consuming. Most metagenomic assays can theoretically detect all pathogens, but often fail to detect viruses because of their small genome and low viral load. Viral metagenomics overcomes this by enrichment of the viral genomic content in a sample. VIDISCA-NGS is one of the available workflows for viral metagenomics, which requires only a small input volume and allows multiplexing of multiple samples per run. The performance of VIDISCA-NGS was tested on 45 cerebrospinal fluid (CSF) samples from patients with suspected CNS infections in which a virus was identified and quantified by polymerase chain reaction. Eighteen were positive for an RNA virus, and 34 for a herpesvirus. VIDISCA-NGS detected all RNA viruses with a viral load >2 × 104 RNA copies/mL (n = 6) and 8 of 12 of the remaining low load samples. Only one herpesvirus was identified by VIDISCA-NGS, however, when withholding a DNase treatment, 11 of 18 samples with a herpesvirus load >104 DNA copies/mL were detected. Our results indicate that VIDISCA-NGS has the capacity to detect low load RNA viruses in CSF. Herpesvirus DNA in clinical samples is probably non-encapsidated and therefore difficult to detect by VIDISCA-NGS.

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Complete genome sequencing of SARS-CoV-2 strains: A pilot survey in Palestine reveals spike mutation H245N

BMC Research Notes ◽

10.1186/s13104-021-05874-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Amer Al-Jawabreh ◽

Suheir Ereqat ◽

Kamal Dumaidi ◽

Hanan Al-Jawabreh ◽

Abedelmajeed Nasereddin

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

Genome Sequencing ◽

Rna Virus ◽

Haplotype Network ◽

Health Concern ◽

Diversity Analysis ◽

High Genetic Diversity ◽

Genetic Diversity Analysis ◽

First Time

Abstract Objectives SARS-CoV-2, severe respiratory syndrome coronavirus-2, is an RNA virus that emerged from China sweeping the globe in the form of a pandemic that became an international public health concern. This pilot study aimed to describe the genetic variation and molecular epidemiology of SARS-CoV-2 in Palestine in fall 2020. Results To achieve these aims, whole genome sequencing of SARS-CoV-2, phylogenetic analysis, haplotype networking and genetic diversity analysis were performed. These analyses revealed a unique spike mutation H245N that has never been reported before. The phylogenetic analysis depicted that three clusters existed in Palestinian SARS-CoV-2 genome sequences, in which cluster-I comprised the majority of clusters by 90%. Congruently, the haplotype network analysis depicted the same three clusters with a total of 39 haplotypes. The genetic diversity analysis showed that Cluster-I is highly diverse as confirmed by statistically significant mutation rate indices, Tajima’s D and Fu-Li’s-F tests (− 2.11 and 2.74, respectively), highest number of mutations (Eta = 120), highest number of haplotypes (h = 17), and highest average number of nucleotide differences between any two sequences (S = 118). The study confirmed the high genetic diversity among the Palestinian of SARS-CoV-2 which possessed high number of mutations including one which was reported for the first time.

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