Coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness

Carbo, Ellen C.; Sidorov, Igor A.; Zevenhoven-Dobbe, Jessika C.; Snijder, Eric J.; Claas, Eric C. J.; Laros, Jeroen F. J.; Kroes, Aloys C.M.; Vries, Jutte J.C. de

doi:10.1016/j.jcv.2020.104594

Cited by 36 publications

(27 citation statements)

References 28 publications

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“…An untargeted sequencing method remains the best strategy for the identification of unknown viral infections, and the genome sequences provide information about the evolutionary history 30 , strain identification 31,32 , and biology of new pathogens 33 . This is evidenced by the recent rapid and impactful metagenomic analysis of SARS-CoV-2 early in the pandemic 34,35 .…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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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Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…There are several approaches used for whole genome sequencing (WGS) of SARS-CoV-2 and can be broadly categorized as targeted and non-targeted i.e., metagenomic approaches (9)(10)(11)(12). Early SARS-CoV-2 genomes were generated using a metagenomic approach given the lack of reference genome at the beginning of the pandemic (9).…”

Section: Introductionmentioning

confidence: 99%

Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol

et al. 2022

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IntroductionThe ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs.MethodsWe utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'.ResultsWe observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60–70% of the genomes could be recovered in samples that had <28% genome coverage with the ARTIC V3 primers. There was no statistically significant (p > 0.05) correlation between Ct value and genome recovery.ConclusionUtilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants.

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“…Decreasing the frequency of wildlife contact with other animals and humans could potentially limit the spread of disease. Regular disease surveillance at these markets and other likely hotspots using whole-genome sequencing and metagenomics could also identify sick animals earlier before a spillover occurs or have the origin or intermediate host known early on to apprise governmental response, where metagenomics has been shown to be able to identify novel coronaviruses and genome sequencing could help direct vaccination efforts [120,121].…”

Section: Future Global Disease Surveillancementioning

confidence: 99%

The Many Faces of Innate Immunity in SARS-CoV-2 Infection

et al. 2021

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The innate immune system is important for initial antiviral response. SARS-CoV-2 can result in overactivity or suppression of the innate immune system. A dysregulated immune response is associated with poor outcomes; with patients having significant Neutrophil-to-Lymphocyte ratios (NLR) due to neutrophilia alongside lymphopenia. Elevated interleukin (IL)-6 and IL-8 leads to overactivity and is a prominent feature of severe COVID-19 patients. IL-6 can result in lymphopenia; where COVID-19 patients typically have significantly altered lymphocyte subsets. IL-8 attracts neutrophils; which may play a significant role in lung tissue damage with the formation of neutrophil extracellular traps leading to cytokine storm or acute respiratory distress syndrome. Several factors like pre-existing co-morbidities, genetic risks, viral pathogenicity, and therapeutic efficacy act as important modifiers of SARS-CoV-2 risks for disease through an interplay with innate host inflammatory responses. In this review, we discuss the role of the innate immune system at play with other important modifiers in SARS-CoV-2 infection.

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Coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness

Cited by 36 publications

References 28 publications

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

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

Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol

The Many Faces of Innate Immunity in SARS-CoV-2 Infection

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