Identification of nsp1 gene as the target of SARS‐CoV‐2 real‐time RT‐PCR using nanopore whole‐genome sequencing

AbstractCOVID-19 global cases have climbed to more than 33 million, with over a million total deaths, as of September, 2020. Real-time massive SARS-CoV-2 whole genome sequencing is key to tracking chains of transmission and estimating the origin of disease outbreaks. Yet no methods have simultaneously achieved high precision, simple workflow, and low cost. We developed a high-precision, cost-efficient SARS-CoV-2 whole genome sequencing platform for COVID-19 genomic surveillance, CorvGenSurv (Coronavirus Genomic Surveillance). CorvGenSurv directly amplified viral RNA from COVID-19 patients’ Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens and sequenced the SARS-CoV-2 whole genome in three segments by long-read, high-throughput sequencing. Sequencing of the whole genome in three segments significantly reduced sequencing data waste, thereby preventing dropouts in genome coverage. We validated the precision of our pipeline by both control genomic RNA sequencing and Sanger sequencing. We produced near full-length whole genome sequences from individuals who were COVID-19 test positive during April to June 2020 in Los Angeles County, California, USA. These sequences were highly diverse in the G clade with nine novel amino acid mutations including NSP12-M755I and ORF8-V117F. With its readily adaptable design, CorvGenSurv grants wide access to genomic surveillance, permitting immediate public health response to sudden threats.

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False COVID-19 cases due to contamination by inactivated virus vaccine

Clinical Infectious Diseases ◽

10.1093/cid/ciab684 ◽

2021 ◽

Author(s):

Kelvin Kai-Wang To ◽

Xin Li ◽

David Christopher Lung ◽

Jonathan Daniel Ip ◽

Wan-Mui Chan ◽

...

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Virus Vaccine ◽

False Positive ◽

Spike Protein ◽

Whole Genome ◽

Rt Pcr ◽

Health Measures ◽

Respiratory Specimens

Abstract A false-positive SARS-CoV-2 RT-PCR result can lead to unnecessary public-health measures. We report two individuals whose respiratory specimens were contaminated by inactivated SARS-CoV-2 vaccine strain(CoronaVac), likely at vaccination premises. Incidentally, whole-genome sequencing of CoronaVac showed adaptive deletions on the spike protein, which do not result in observable changes of antigenicity.

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Genome Sequence ofListeria monocytogenesStrain HPB2088 (Serotype 1/2a), an Environmental Isolate Collected in Canada in 1994

Genome Announcements ◽

10.1128/genomea.00760-16 ◽

2016 ◽

Vol 4 (4) ◽

Cited By ~ 1

Author(s):

Arthur W. Pightling ◽

Hugh Rand ◽

Errol Strain ◽

Franco Pagotto

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Genome Sequence ◽

Foodborne Pathogen ◽

Environmental Isolate ◽

Severe Illness ◽

Whole Genome ◽

Serotype 1

Listeria monocytogenesis a foodborne pathogen that causes severe illness. Thus, ongoing efforts at real-time whole-genome sequencing are of utmost importance. However, it is also important that retrospective analyses that place these data into context be performed. Here, we present the genome sequence of strain HPB2088, which was collected in 1994.

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Real-Time Whole-Genome Sequencing for Surveillance ofListeria monocytogenes, France

Emerging Infectious Diseases ◽

10.3201/eid2309.170336 ◽

2017 ◽

Vol 23 (9) ◽

pp. 1462-1470 ◽

Cited By ~ 67

Author(s):

Alexandra Moura ◽

Mathieu Tourdjman ◽

Alexandre Leclercq ◽

Estelle Hamelin ◽

Edith Laurent ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Whole Genome ◽

Oflisteria Monocytogenes

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Real time, field-deployable whole genome sequencing of malaria parasites using nanopore technology

10.1101/2020.12.17.423341 ◽

2020 ◽

Author(s):

Zahra Razook ◽

Somya Mehra ◽

Brittany Gilchrist ◽

Digjaya Utama ◽

Dulcie Lautu-Gumal ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Error Rates ◽

Whole Genome ◽

Nanopore Sequencing ◽

Virulence Determinants ◽

Malaria Parasites ◽

Additional Information ◽

Natural Isolates

ABSTRACTMalaria parasite genomes have been generated predominantly using short read sequencing technology which can be slow, requires advanced laboratory training and does not adequately interrogate complex genomic regions that harbour important malaria virulence determinants. The portable Oxford Nanopore Technologies MinION platform generates long reads in real time and may overcome these limitations. We present compelling evidence that Nanopore sequencing delivers valuable additional information for malaria parasites with comparable data fidelity for single nucleotide variant (SNV) calls, compared to standard Illumina whole genome sequencing. We demonstrate this through sequencing of pure Plasmodium falciparum DNA, mock infections and natural isolates. Nanopore has low error rates for haploid SNV genotyping and identifies structural variants (SVs) not detected with short reads. Nanopore genomes are directly comparable to publically available genomes and produce high quality end to end chromosome assemblies. Nanopore sequencing will expedite genomic surveillance of malaria and provide new insights into parasite genome biology.

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Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

10.1101/2020.02.20.958728 ◽

2020 ◽

Author(s):

AG Stewart ◽

EP Price ◽

K Schabacker ◽

M Birikmen ◽

PNA Harris ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Real Time Pcr ◽

Generation Cephalosporin ◽

Whole Genome ◽

Third Generation ◽

Sensitivity Testing

AbstractThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 258 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using a rapid (~40 min) pentaplex real-time PCR assay targeting the most common extended spectrum β-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). Additionally, AmpC CMY β-lactamase prevalence was examined using a singleplex PCR. A subset of isolates, including all 3GC-R isolates obtained from the intensive care unit, were subjected to whole-genome sequencing (WGS) to assess transmission dynamics, the presence of unidentified resistance determinants, and genotyping accuracy. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance was identified in 97% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, the majority (98.3%) of isolates harbored at least one β-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. WGS of β-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. No evidence of transmission among intensive care unit patients was identified. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

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AmpliCoV: Rapid Whole-Genome Sequencing Using Multiplex PCR Amplification and Real-Time Oxford Nanopore MinION Sequencing Enables Rapid Variant Identification of SARS-CoV-2

Frontiers in Microbiology ◽

10.3389/fmicb.2021.651151 ◽

2021 ◽

Vol 12 ◽

Author(s):

Annika Brinkmann ◽

Sophie-Luisa Ulm ◽

Steven Uddin ◽

Sophie Förster ◽

Dominique Seifert ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Phylogenetic Analyses ◽

Diagnostic Methods ◽

Virus Concentration ◽

Sequence Information ◽

Whole Genome ◽

Oxford Nanopore ◽

A Genome

Since the emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the scientific community has been sharing data on epidemiology, diagnostic methods, and whole-genomic sequences almost in real time. The latter have already facilitated phylogenetic analyses, transmission chain tracking, protein modeling, the identification of possible therapeutic targets, timely risk assessment, and identification of novel variants. We have established and evaluated an amplification-based approach for whole-genome sequencing of SARS-CoV-2. It can be used on the miniature-sized and field-deployable sequencing device Oxford Nanopore MinION, with sequencing library preparation time of 10 min. We show that the generation of 50,000 total reads per sample is sufficient for a near complete coverage (>90%) of the SARS-CoV-2 genome directly from patient samples even if virus concentration is low (Ct 35, corresponding to approximately 5 genome copies per reaction). For patient samples with high viral load (Ct 18–24), generation of 50,000 reads in 1–2 h was shown to be sufficient for a genome coverage of >90%. Comparison to Illumina data reveals an accuracy that suffices to identify virus mutants. AmpliCoV can be applied whenever sequence information on SARS-CoV-2 is required rapidly, for instance for the identification of circulating virus mutants.

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Detection of SARS-CoV-2 from Patient Fecal Samples by Whole Genome Sequencing

10.21203/rs.3.rs-48863/v1 ◽

2020 ◽

Author(s):

Andreas Papoutsis ◽

Thomas Borody ◽

Siba Dolai ◽

Jordan Daniels ◽

Skylar Steinberg ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Analysis ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Rt Pcr ◽

Whole Genome Analysis ◽

Fecal Samples ◽

Oral Transmission ◽

Study Participants

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment NGS from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal-oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication.

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