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  1. Article ; Online: Targeted genomic sequencing of avian influenza viruses in wetland sediment from wild bird habitats.

    Kuchinski, Kevin S / Coombe, Michelle / Mansour, Sarah C / Cortez, Gabrielle Angelo P / Kalhor, Marzieh / Himsworth, Chelsea G / Prystajecky, Natalie A

    Applied and environmental microbiology

    2024  Volume 90, Issue 2, Page(s) e0084223

    Abstract: Diverse influenza A viruses (IAVs) circulate in wild birds, including highly pathogenic strains that infect poultry and humans. Consequently, surveillance of IAVs in wild birds is a cornerstone of agricultural biosecurity and pandemic preparedness. ... ...

    Abstract Diverse influenza A viruses (IAVs) circulate in wild birds, including highly pathogenic strains that infect poultry and humans. Consequently, surveillance of IAVs in wild birds is a cornerstone of agricultural biosecurity and pandemic preparedness. Surveillance is traditionally done by testing wild birds directly, but obtaining these specimens is labor intensive, detection rates can be low, and sampling is often biased toward certain avian species. As a result, local incursions of dangerous IAVs are rarely detected before outbreaks begin. Testing environmental specimens from wild bird habitats has been proposed as an alternative surveillance strategy. These specimens are thought to contain diverse IAVs deposited by a broad range of avian hosts, including species that are not typically sampled by surveillance programs. To enable this surveillance strategy, we developed a targeted genomic sequencing method for characterizing IAVs in these challenging environmental specimens. It combines custom hybridization probes, unique molecular index-based library construction, and purpose-built bioinformatic tools, allowing IAV genomic material to be enriched and analyzed with single-fragment resolution. We demonstrated our method on 90 sediment specimens from wetlands around Vancouver, Canada. We recovered 2,312 IAV genome fragments originating from all eight IAV genome segments. Eleven hemagglutinin subtypes and nine neuraminidase subtypes were detected, including H5, the current global surveillance priority. Our results demonstrate that targeted genomic sequencing of environmental specimens from wild bird habitats could become a valuable complement to avian influenza surveillance programs.IMPORTANCEIn this study, we developed genome sequencing tools for characterizing avian influenza viruses in sediment from wild bird habitats. These tools enable an environment-based approach to avian influenza surveillance. This could improve early detection of dangerous strains in local wild birds, allowing poultry producers to better protect their flocks and prevent human exposures to potential pandemic threats. Furthermore, we purposefully developed these methods to contend with viral genomic material that is diluted, fragmented, incomplete, and derived from multiple strains and hosts. These challenges are common to many environmental specimens, making these methods broadly applicable for genomic pathogen surveillance in diverse contexts.
    MeSH term(s) Animals ; Animals, Wild ; Birds ; Genomics ; Influenza A virus/genetics ; Influenza in Birds/epidemiology ; Phylogeny ; Poultry ; Wetlands
    Language English
    Publishing date 2024-01-23
    Publishing country United States
    Document type Journal Article
    ZDB-ID 223011-2
    ISSN 1098-5336 ; 0099-2240
    ISSN (online) 1098-5336
    ISSN 0099-2240
    DOI 10.1128/aem.00842-23
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline.

    Kuchinski, Kevin S / Jassem, Agatha N / Prystajecky, Natalie A

    Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology

    2020  Volume 131, Page(s) 104581

    Abstract: Introduction: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing ... ...

    Abstract Introduction: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans.
    Methods: We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays.
    Results: For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed.
    Discussion: Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.
    MeSH term(s) Betacoronavirus/genetics ; COVID-19 ; COVID-19 Testing ; Clinical Laboratory Techniques/methods ; Computational Biology ; Computer Simulation ; Coronavirus Infections/diagnosis ; Coronavirus Infections/virology ; Genome, Viral ; Humans ; Oligonucleotides/genetics ; Pandemics ; Pneumonia, Viral/diagnosis ; Pneumonia, Viral/virology ; RNA, Viral ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Sensitivity and Specificity
    Chemical Substances Oligonucleotides ; RNA, Viral
    Keywords covid19
    Language English
    Publishing date 2020-08-21
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 1446080-4
    ISSN 1873-5967 ; 1386-6532
    ISSN (online) 1873-5967
    ISSN 1386-6532
    DOI 10.1016/j.jcv.2020.104581
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: ProbeTools: designing hybridization probes for targeted genomic sequencing of diverse and hypervariable viral taxa.

    Kuchinski, Kevin S / Duan, Jun / Himsworth, Chelsea / Hsiao, William / Prystajecky, Natalie A

    BMC genomics

    2022  Volume 23, Issue 1, Page(s) 579

    Abstract: Background: Sequencing viruses in many specimens is hindered by excessive background material from hosts, microbiota, and environmental organisms. Consequently, enrichment of target genomic material is necessary for practical high-throughput viral ... ...

    Abstract Background: Sequencing viruses in many specimens is hindered by excessive background material from hosts, microbiota, and environmental organisms. Consequently, enrichment of target genomic material is necessary for practical high-throughput viral genome sequencing. Hybridization probes are widely used for enrichment in many fields, but their application to viral sequencing faces a major obstacle: it is difficult to design panels of probe oligo sequences that broadly target many viral taxa due to their rapid evolution, extensive diversity, and genetic hypervariability. To address this challenge, we created ProbeTools, a package of bioinformatic tools for generating effective viral capture panels, and for assessing coverage of target sequences by probe panel designs in silico. In this study, we validated ProbeTools by designing a panel of 3600 probes for subtyping the hypervariable haemagglutinin (HA) and neuraminidase (NA) genome segments of avian-origin influenza A viruses (AIVs). Using in silico assessment of AIV reference sequences and in vitro capture on egg-cultured viral isolates, we demonstrated effective performance by our custom AIV panel and ProbeTools' suitability for challenging viral probe design applications.
    Results: Based on ProbeTool's in silico analysis, our panel provided broadly inclusive coverage of 14,772 HA and 11,967 NA reference sequences. For each reference sequence, we calculated the percentage of nucleotide positions covered by our panel in silico; 90% of HA and NA references sequences had at least 90.8 and 95.1% of their nucleotide positions covered respectively. We also observed effective in vitro capture on a representative collection of 23 egg-cultured AIVs that included isolates from wild birds, poultry, and humans and representatives from all HA and NA subtypes. Forty-two of forty-six HA and NA segments had over 98.3% of their nucleotide positions significantly enriched by our custom panel. These in vitro results were further used to validate ProbeTools' in silico coverage assessment algorithm; 89.2% of in silico predictions were concordant with in vitro results.
    Conclusions: ProbeTools generated an effective panel for subtyping AIVs that can be deployed for genomic surveillance, outbreak prevention, and pandemic preparedness. Effective probe design against hypervariable AIV targets also validated ProbeTools' design and coverage assessment algorithms, demonstrating their suitability for other challenging viral capture applications.
    MeSH term(s) Animals ; Genomics ; Humans ; Influenza A virus ; Influenza in Birds/genetics ; Neuraminidase/genetics ; Nucleotides ; Phylogeny
    Chemical Substances Nucleotides ; Neuraminidase (EC 3.2.1.18)
    Language English
    Publishing date 2022-08-12
    Publishing country England
    Document type Journal Article
    ZDB-ID 2041499-7
    ISSN 1471-2164 ; 1471-2164
    ISSN (online) 1471-2164
    ISSN 1471-2164
    DOI 10.1186/s12864-022-08790-4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Nasopharyngeal angiotensin converting enzyme 2 (ACE2) expression as a risk-factor for SARS-CoV-2 transmission in concurrent hospital associated outbreaks.

    Nikiforuk, Aidan M / Kuchinski, Kevin S / Short, Katy / Roman, Susan / Irvine, Mike A / Prystajecky, Natalie / Jassem, Agatha N / Patrick, David M / Sekirov, Inna

    BMC infectious diseases

    2024  Volume 24, Issue 1, Page(s) 262

    Abstract: Background: Widespread human-to-human transmission of the severe acute respiratory syndrome coronavirus two (SARS-CoV-2) stems from a strong affinity for the cellular receptor angiotensin converting enzyme two (ACE2). We investigate the relationship ... ...

    Abstract Background: Widespread human-to-human transmission of the severe acute respiratory syndrome coronavirus two (SARS-CoV-2) stems from a strong affinity for the cellular receptor angiotensin converting enzyme two (ACE2). We investigate the relationship between a patient's nasopharyngeal ACE2 transcription and secondary transmission within a series of concurrent hospital associated SARS-CoV-2 outbreaks in British Columbia, Canada.
    Methods: Epidemiological case data from the outbreak investigations was merged with public health laboratory records and viral lineage calls, from whole genome sequencing, to reconstruct the concurrent outbreaks using infection tracing transmission network analysis. ACE2 transcription and RNA viral load were measured by quantitative real-time polymerase chain reaction. The transmission network was resolved to calculate the number of potential secondary cases. Bivariate and multivariable analyses using Poisson and Negative Binomial regression models was performed to estimate the association between ACE2 transcription the number of SARS-CoV-2 secondary cases.
    Results: The infection tracing transmission network provided n = 76 potential transmission events across n = 103 cases. Bivariate comparisons found that on average ACE2 transcription did not differ between patients and healthcare workers (P = 0.86). High ACE2 transcription was observed in 98.6% of transmission events, either the primary or secondary case had above average ACE2. Multivariable analysis found that the association between ACE2 transcription (log
    Conclusion: Our study suggests that ACE2 transcription has a positive association with SARS-CoV-2 secondary transmission in admitted inpatients, but not health care workers in concurrent hospital associated outbreaks, and it should be further investigated as a risk-factor for viral transmission.
    MeSH term(s) Humans ; Angiotensin-Converting Enzyme 2 ; British Columbia/epidemiology ; COVID-19/epidemiology ; Disease Outbreaks ; Hospitals ; RNA ; SARS-CoV-2/genetics
    Chemical Substances Angiotensin-Converting Enzyme 2 (EC 3.4.17.23) ; RNA (63231-63-0) ; ACE2 protein, human (EC 3.4.17.23)
    Language English
    Publishing date 2024-02-26
    Publishing country England
    Document type Journal Article
    ZDB-ID 2041550-3
    ISSN 1471-2334 ; 1471-2334
    ISSN (online) 1471-2334
    ISSN 1471-2334
    DOI 10.1186/s12879-024-09067-9
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline

    Kuchinski, Kevin S. / Jassem, Agatha N. / Prystajecky, Natalie A.

    Journal of Clinical Virology

    2020  Volume 131, Page(s) 104581

    Keywords Virology ; Infectious Diseases ; covid19
    Language English
    Publisher Elsevier BV
    Publishing country us
    Document type Article ; Online
    ZDB-ID 1446080-4
    ISSN 1873-5967 ; 1386-6532
    ISSN (online) 1873-5967
    ISSN 1386-6532
    DOI 10.1016/j.jcv.2020.104581
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article: Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline

    Kuchinski, Kevin S / Jassem, Agatha N / Prystajecky, Natalie A

    J Clin Virol

    Abstract: INTRODUCTION: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing ... ...

    Abstract INTRODUCTION: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans. METHODS: We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays. RESULTS: For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed. DISCUSSION: Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.
    Keywords covid19
    Publisher WHO
    Document type Article
    Note WHO #Covidence: #726607
    Database COVID19

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  7. Article ; Online: Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens.

    Kuchinski, Kevin S / Nguyen, Jason / Lee, Tracy D / Hickman, Rebecca / Jassem, Agatha N / Hoang, Linda M N / Prystajecky, Natalie A / Tyson, John R

    International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases

    2021  Volume 114, Page(s) 51–54

    Abstract: Mutations in emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages can interfere with laboratory methods used to generate viral genome sequences for public health surveillance. We identified 20 mutations that are widespread in ... ...

    Abstract Mutations in emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages can interfere with laboratory methods used to generate viral genome sequences for public health surveillance. We identified 20 mutations that are widespread in variant of concern lineages and affect widely used sequencing protocols by the ARTIC network and Freed et al. Three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada. We provide laboratory validation of protocol modifications that restored sequencing performance. The study findings indicate that genomic sequencing protocols require immediate updating to address emerging mutations. This work also suggests that routine monitoring and protocol updates will be necessary as SARS-CoV-2 continues to evolve. The bioinformatic and laboratory approaches used here provide guidance for this kind of assay maintenance.
    MeSH term(s) British Columbia ; COVID-19 ; Genome, Viral/genetics ; Genomics ; Humans ; Mutation ; SARS-CoV-2
    Language English
    Publishing date 2021-10-29
    Publishing country Canada
    Document type Journal Article
    ZDB-ID 1331197-9
    ISSN 1878-3511 ; 1201-9712
    ISSN (online) 1878-3511
    ISSN 1201-9712
    DOI 10.1016/j.ijid.2021.10.050
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  8. Article ; Online: Rapid initiation of nasal saline irrigation to reduce severity in high-risk COVID+ outpatients.

    Baxter, Amy L / Schwartz, Kyle R / Johnson, Ryan W / Kuchinski, Ann-Marie / Swartout, Kevin M / Srinivasa Rao, Arni S R / Gibson, Robert W / Cherian, Erica / Giller, Taylor / Boomer, Houlton / Lyon, Matthew / Schwartz, Richard

    Ear, nose, & throat journal

    2022  , Page(s) 1455613221123737

    Abstract: Objective: To determine whether initiating saline nasal irrigation after COVID-19 diagnosis reduces hospitalization and death in high-risk outpatients compared with observational controls, and if irrigant composition impacts severity.: Methods: ... ...

    Abstract Objective: To determine whether initiating saline nasal irrigation after COVID-19 diagnosis reduces hospitalization and death in high-risk outpatients compared with observational controls, and if irrigant composition impacts severity.
    Methods: Participants 55 and older were enrolled within 24 hours of a + PCR COVID-19 test between September 24 and December 21, 2020. Among 826 screened, 79 participants were enrolled and randomly assigned to add 2.5 mL povidone-iodine 10% or 2.5 mL sodium bicarbonate to 240 mL of isotonic nasal irrigation twice daily for 14 days. The primary outcome was hospitalization or death from COVID-19 within 28 days of enrollment by daily self-report confirmed with phone calls and hospital records, compared to the CDC Surveillance Dataset covering the same time. Secondary outcomes compared symptom resolution by irrigant additive.
    Results: Seventy-nine high-risk participants were enrolled (mean [SD] age, 64 [8] years; 36 [46%] women; 71% Non-Hispanic White), with mean BMI 30.3. Analyzed by intention-to-treat, by day 28, COVID-19 symptoms resulted in one ED visit and no hospitalizations in 42 irrigating with alkalinization, one hospitalization of 37 in the povidone-iodine group, (1.27%) and no deaths. Of nearly three million CDC cases, 9.47% were known to be hospitalized, with an additional 1.5% mortality in those without hospitalization data. Age, sex, and percentage with pre-existing conditions did not significantly differ by exact binomial test from the CDC dataset, while reported race and hospitalization rate did. The total risk of hospitalization or death (11%) was 8.57 times that of enrolled nasal irrigation participants (SE = 2.74;
    Conclusion: SARS-CoV-2+ participants initiating nasal irrigation were over 8 times less likely to be hospitalized than the national rate.
    Language English
    Publishing date 2022-08-25
    Publishing country United States
    Document type Journal Article
    ZDB-ID 750153-5
    ISSN 1942-7522 ; 0145-5613
    ISSN (online) 1942-7522
    ISSN 0145-5613
    DOI 10.1177/01455613221123737
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  9. Article ; Online: Nasopharyngeal Angiotensin Converting Enzyme 2 (ACE2) Expression as a Risk-Factor for SARS-CoV-2 Transmission in Concurrent Hospital Associated Outbreaks

    Nikiforuk, Aidan M / Kuchinski, Kevin S / Short, Katy / Roman, Susan / Irvine, Michael A / Prystajecky, Natalie / Jassem, Agatha N / Patrick, David M / Sekirov, Inna

    medRxiv

    Abstract: Background: Widespread human-to-human transmission of the severe acute respiratory syndrome coronavirus two (SARS-CoV-2) stems from a strong affinity for the cellular receptor angiotensin converting enzyme two (ACE2). We investigate the relationship ... ...

    Abstract Background: Widespread human-to-human transmission of the severe acute respiratory syndrome coronavirus two (SARS-CoV-2) stems from a strong affinity for the cellular receptor angiotensin converting enzyme two (ACE2). We investigate the relationship between a patient9s nasopharyngeal ACE2 transcription and secondary transmission within a series of concurrent hospital associated SARS-CoV-2 outbreaks in British Columbia, Canada. Methods: Epidemiological case data from the outbreak investigations was merged with public health laboratory records and viral lineage calls, from whole genome sequencing, to reconstruct the concurrent outbreaks using infection tracing transmission network analysis. ACE2 transcription and RNA viral load were measured by quantitative real-time polymerase chain reaction. The transmission network was resolved to calculate the number of potential secondary cases. Bivariate and multivariable analyses using Poisson and Negative Binomial regression models was performed to estimate the association between ACE2 transcription the number of SARS-CoV-2 secondary cases. Results: The infection tracing transmission network provided n = 76 potential transmission events across n = 103 cases. Bivariate comparisons found that on average ACE2 transcription did not differ between patients and healthcare workers (P = 0.86). High ACE2 transcription was observed in 98.6% of transmission events, either the primary or secondary case had above average ACE2. Multivariable analysis found that the association between ACE2 transcription and the number of secondary transmission events differs between patients and healthcare workers. In health care workers Negative Binomial regression estimated that a one unit change in ACE2 transcription decreases the number of secondary cases (B = -0.132 (95%CI: -0.255 to -0.0181) adjusting for RNA viral load. Conversely, in patients a one unit change in ACE2 transcription increases the number of secondary cases (B = 0.187 (95% CI: 0.0101 to 0.370) adjusting for RNA viral load. Sensitivity analysis found no significant relationship between ACE2 and secondary transmission in health care workers and confirmed the positive association among patients. Conclusion: Our study suggests that ACE2 transcription has a positive association with SARS-CoV-2 secondary transmission in admitted inpatients, but not health care workers in concurrent hospital associated outbreaks, and it should be further investigated as a risk-factor for viral transmission.
    Keywords covid19
    Language English
    Publishing date 2023-03-22
    Publisher Cold Spring Harbor Laboratory Press
    Document type Article ; Online
    DOI 10.1101/2023.03.20.23287264
    Database COVID19

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  10. Article ; Online: Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens

    Kuchinski, Kevin S / Nguyen, Jason / Lee, Tracy D / Hickman, Rebecca / Jassem, Agatha N / Hoang, Linda MN / Prystajecky, Natalie A / Tyson, John R

    medRxiv

    Abstract: Mutations in emerging SARS-CoV-2 lineages can interfere with the laboratory methods used to generate high-quality genome sequences for COVID-19 surveillance. Here, we identify 46 mutations in current variant of concern lineages affecting the widely used ... ...

    Abstract Mutations in emerging SARS-CoV-2 lineages can interfere with the laboratory methods used to generate high-quality genome sequences for COVID-19 surveillance. Here, we identify 46 mutations in current variant of concern lineages affecting the widely used laboratory protocols for SARS-CoV-2 genomic sequencing by Freed et al. and the ARTIC network. We provide laboratory data showing how three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada, and we also demonstrate how we modified the Freed et al. protocol to restore performance.
    Keywords covid19
    Language English
    Publishing date 2021-06-04
    Publisher Cold Spring Harbor Laboratory Press
    Document type Article ; Online
    DOI 10.1101/2021.06.01.21258181
    Database COVID19

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