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• W4285807885 abstract "Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of CT values (11.3 to 36.7; median, 21.6). Of 428 positive samples, 413 (96.5%) generated genomes with <10% unknown bases, with a mean genome coverage of 13,545× ± SD 8382×. No genomes were recovered from PCR-negative specimens (n = 30) or from specimens positive for non–SARS-CoV-2 respiratory viruses (n = 20). Compared with whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks, with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control. Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of CT values (11.3 to 36.7; median, 21.6). Of 428 positive samples, 413 (96.5%) generated genomes with <10% unknown bases, with a mean genome coverage of 13,545× ± SD 8382×. No genomes were recovered from PCR-negative specimens (n = 30) or from specimens positive for non–SARS-CoV-2 respiratory viruses (n = 20). Compared with whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks, with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control. Since the deposition of the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole genome sequence (NC_045512.1) in January 2020, >4 million SARS-CoV-2 genomes have been deposited to public data repositories, far exceeding any other human pathogen.1Elbe S. Buckland-Merrett G. Data, disease and diplomacy: GISAID’s innovative contribution to global health.Glob Chall. 2017; 1: 33-46Crossref PubMed Google Scholar,2Moustafa A.M. Planet P.J. Jumping a moving train: SARS-CoV-2 evolution in real time.J Pediatr Infect Dis Soc. 2021; 10: S96-S105Crossref PubMed Scopus (3) Google Scholar Such a feat has been made possible because of advances in next-generation sequencing (NGS) technologies that enable near real-time genomic surveillance.3Paden C.R. Tao Y. Queen K. Zhang J. Li Y. Uehara A. 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Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization.Cell Host Microbe. 2021; 29: 477-488.e4Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar and viral sequencing can be used to monitor the accumulation of mutations during long-term viral replication in immunocompromised individuals.30Choi B. Choudhary M.C. Regan J. Sparks J.A. Padera R.F. Qiu X. Solomon I.H. Kuo H.-H. Boucau J. Bowman K. Adhikari U.D. Winkler M.L. Mueller A.A. Hsu T.Y.-T. Desjardins M. Baden L.R. Chan B.T. Walker B.D. Lichterfeld M. Brigl M. Kwon D.S. Kanjilal S. Richardson E.T. Jonsson A.H. Alter G. Barczak A.K. Hanage W.P. Yu X.G. Gaiha G.D. Seaman M.S. Cernadas M. Li J.Z. Persistence and evolution of SARS-CoV-2 in an immunocompromised host.N Engl J Med. 2020; 383: 2291-2293Crossref PubMed Scopus (487) Google Scholar As treatment regimens expand, validated WGS assays are needed not only for genomic surveillance but also for high-quality, clinically actionable data with rapid turnaround times.Multiplexed amplicon sequencing methods have proven to be faster, more sensitive, and more cost-effective than shotgun and capture-based approaches, enabling genome recovery across a wide range of viral loads.31Xiao M. Liu X. Ji J. Li M. Li J. Yang L. Sun W. Ren P. Yang G. Zhao J. Liang T. Ren H. Chen T. Zhong H. Song W. Wang Y. Deng Z. Zhao Y. Ou Z. Wang D. Cai J. Cheng X. Feng T. Wu H. Gong Y. Yang H. Wang J. Xu X. Zhu S. Chen F. Zhang Y. Chen W. Li Y. Li J. Multiple approaches for massively parallel sequencing of SARS-CoV-2 genomes directly from clinical samples.Genome Med. 2020; 12: 57Crossref PubMed Scopus (60) Google Scholar,32Charre C. Ginevra C. Sabatier M. Regue H. Destras G. Brun S. Burfin G. Scholtes C. Morfin F. Valette M. Lina B. Bal A. Josset L. Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation.Virus Evol. 2020; 6veaa075Crossref Scopus (58) Google Scholar We previously tested one such panel from Swift Biosciences and demonstrated genome recovery up to a CT of 36 across a broad range of isolates.33Addetia A. Lin M.J. Peddu V. Roychoudhury P. Jerome K.R. Greninger A.L. Sensitive recovery of complete SARS-CoV-2 genomes from clinical samples by use of Swift Biosciences’ SARS-CoV-2 multiplex amplicon sequencing panel.J Clin Microbiol. 2020; 59: e02226-20Crossref Scopus (28) Google Scholar Designed against the SARS-CoV-2 Wuhan–Hu-1 complete genome (https://www.ncbi.nlm.nih.gov/nuccore, accession number NC_045512.2, last accessed July 12, 2022), the Swift Normalase Amplicon Panel (SNAP) primer set amplifies 345 amplicons ranging from 116 to 255 bp (average, 150 bp) in a single tube to cover the approximately 30-kb SARS-CoV-2 genome. This assay can generate libraries in <3 hours using an input concentration of only 10 to 100+ viral copies for single-strand cDNA or double-strand cDNA synthesis. This can be followed by either manual normalization or Swift Biosciences' proprietary enzymatic normalization of multiplexed libraries for equimolar pools.For clinical use, sequencing assays need to be rigorously validated, documented, and performed in Clinical Laboratory Improvement Amendments–accredited laboratories. However, no specific guidelines currently exist for the development and validation of WGS assays for SARS-CoV-2. We validated the Swift Biosciences' one-tube SARS-CoV-2 SNAP Version 2.0—the first clinical validation of an NGS-based assay for WGS of SARS-CoV-2 to our knowledge—according to US Food and Drug Administration’s “Considerations for Design, Development, and Analytical Validation of Next Generation Sequencing-Based in Vitro Diagnostics Intended to Aid in the Diagnosis of Suspected Germline Diseases” (https://www.fda.gov/media/99208/download, last accessed January 4, 2021) and US Food and Drug Administration's “Submitting Next Generation Sequencing Data to the Division of Antiviral Products Guidance for Industry” Technical Specifications Document (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/submitting-next-generation-sequencing-data-division-antiviral-products-guidance-industry-technical, last accessed August 19, 2021). Using clinical specimens, the analytical sensitivity, analytical specificity, limit of detection, accuracy, and precision (reproducibility and repeatability) of the assay were evaluated, establishing acceptance criteria for sequencing libraries and output genomes. This assay is now available as a clinically orderable test, with results returned to physicians to aid in disease management and treatment. It has been used in multiple vaccine trials, research studies, validation of other NGS-based assays, and sequencing SARS-CoV-2 for public health surveillance and outbreak investigation.4Fauver J.R. Petrone M.E. Hodcroft E.B. Shioda K. Ehrlich H.Y. Watts A.G. et al.Coast-to-coast spread of SARS-CoV-2 during the early epidemic in the United States.Cell. 2020; 181: 990-996.e5Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar,6Bedford T. Greninger A.L. Roychoudhury P. Starita L.M. Famulare M. Huang M.-L. et al.Cryptic transmission of SARS-CoV-2 in Washington state.Science. 2020; 370: 571-575Crossref PubMed Google Scholar,10Lieberman N.A.P. Peddu V. Xie H. Shrestha L. Huang M.-L. Mears M.C. Cajimat M.N. Bente D.A. Shi P.-Y. Bovier F. Roychoudhury P. Jerome K.R. Moscona A. Porotto M. Greninger A.L. In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age.PLOS Biol. 2020; 18e3000849Crossref PubMed Google Scholar,34Sadoff J. le Gars M. Shukarev G. Heerwegh D. Truyers C. de Groot A.M. Stoop J. Tete S. van Damme W. Leroux-Roels I. Berghmans P.-J. Kimmel M. van Damme P. de Hoon J. Smith W. Stephenson K.E. de Rosa S.C. Cohen K.W. McElrath M.J. Cormier E. Scheper G. Barouch D.H. Hendriks J. Struyf F. Douoguih M. van Hoof J. Schuitemaker H. Interim results of a phase 1–2a trial of Ad26.COV2.S Covid-19 vaccine.N Engl J Med. 2021; 384: 1824-1835Crossref PubMed Scopus (606) Google Scholar, 35Sadoff J. Gray G. Vandebosch A. Cárdenas V. Shukarev G. Grinsztejn B. Goepfert P.A. Truyers C. Fennema H. Spiessens B. Offergeld K. Scheper G. Taylor K.L. Robb M.L. Treanor J. Barouch D.H. Stoddard J. Ryser M.F. Marovich M.A. Neuzil K.M. Corey L. Cauwenberghs N. Tanner T. Hardt K. Ruiz-Guiñazú J. le Gars M. Schuitemaker H. van Hoof J. Struyf F. Douoguih M. Safety and efficacy of single-dose Ad26.COV2.S vaccine against Covid-19.N Engl J Med. 2021; 384: 2187-2201Crossref PubMed Scopus (965) Google Scholar, 36Hilt E.E. Boocock J. Trejo M. Le C.Q. Guo L. Zhang Y. Sathe L. Arboleda V.A. Yin Y. Bloom J.S. Wang P.-C. Elmore J.G. Kruglyak L. Shrestha L. Bakhash S.A.M. Lin M. Xie H. Huang M.-L. Roychoudhury P. Greninger A. Chandrasekaran S. Yang S. Garner O.B. Retrospective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in symptomatic patients prior to widespread diagnostic testing in southern California.Clin Infect Dis. 2022; 74: 271-277Crossref PubMed Scopus (2) Google Scholar, 37McEwen A.E. Cohen S. Bryson-Cahn C. Liu C. Pergam S.A. Lynch J. Schippers A. Strand K. Whimbey E. Mani N.S. Zelikoff A.J. Makarewicz V.A. Brown E.R. Bakhash S.A.M. Baker N.R. Castor J. Livingston R.J. Huang M.-L. Jerome K.R. Greninger A.L. Roychoudhury P. Variants of concern are overrepresented among postvaccination breakthrough infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Washington state.Clin Infect Dis. 2022; 74: 1089-1092Crossref PubMed Scopus (16) Google Scholar, 38Lin M.J. Rachleff V.M. Xie H. Shrestha L. Lieberman N.A.P. Peddu V. Addetia A. Casto A.M. Breit N. Mathias P.C. Huang M.-L. Jerome K.R. Greninger A.L. Roychoudhury P. Host–pathogen dynamics in longitudinal clinical specimens from patients with COVID-19.Sci Rep. 2022; 12: 5856Crossref PubMed Scopus (2) Google ScholarMaterials and MethodsPerformance EvaluationExisting US Food and Drug Administration guidelines were used for designing, developing, and establishing the analytical validity of NGS-based tests for the diagnosis of suspected germline diseases and development of antivirals to clinically validate a multiplex amplicon sequencing method for WGS of SARS-CoV-2 (Supplemental Figure S1). The aim was to determine analytical sensitivity (limit of detection), analytical specificity, accuracy, and precision (reproducibility and repeatability) of the assay using remnant clinical specimens described below.Clinical SpecimensUse of deidentified remnant clinical specimens from University of Washington Virology Laboratory for SARS-CoV-2 testing was approved by the University of Washington Institutional Review Board. Specimens were tested for SARS-CoV-2 using one of the following PCR assays: CDC-based Laboratory Developed Assays, Abbott m2000 (Abbott Laboratories, Chicago, IL); Roche Cobas (Roche, Basel, Switzerland); or Hologic Panther/Panther Fusion (Hologic, Marlborough, MA).39Lieberman J.A. Pepper G. Naccache S.N. Huang M.-L. Jerome K.R. Greninger A.L. Comparison of commercially available and laboratory-developed assays for in vitro detection of SARS-CoV-2 in clinical laboratories.J Clin Microbiol. 2021; 58: e00821-20Crossref Scopus (156) Google Scholar,40Degli-Angeli E. Dragavon J. Huang M.-L. Lucic D. Cloherty G. Jerome K.R. Greninger A.L. Coombs R.W. Validation and verification of the Abbott RealTime SARS-CoV-2 assay analytical and clinical performance.J Clin Virol. 2020; 129: 104474Crossref PubMed Scopus (32) Google Scholar Samples used for analysis of specificity were tested using University of Washington Virology Laboratory respiratory virus panel to identify non–SARS-CoV-2 respiratory pathogens, including respiratory syncytial virus, influenza virus type A, parainfluenza virus types 1, 2, and 3, and human metapneumovirus.41Kuypers J. Wright N. Ferrenberg J. Huang M.-L. Cent A. Corey L. Morrow R. Comparison of real-time PCR assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children.J Clin Microbiol. 2006; 44: 2382-2388Crossref PubMed Scopus (260) Google ScholarLaboratory-confirmed specimens used in this study came from nasal or nasopharyngeal swabs collected in either phosphate-buffered saline or viral transport medium that had >500 μL volume remaining. This included SARS-CoV-2–positive specimens (n = 428), specimens negative for both SARS-CoV-2 and other respiratory viruses (n = 30), and specimens positive only for other respiratory viruses (n = 20). Water was used as a negative control, and previously confirmed and sequenced SARS-CoV-2–positive specimens were used as positive controls.SARS-CoV-2 Virus Culture IsolatesFor detection of within-sample variation, two previously sequenced culture isolates from different clades were used: WA-UW-20236 TM, EPI_ISL_4926371, Nextstrain clade 20A, and WA-UW-19433 TM, EPI_ISL_4926374, 20B. The culture isolates had a viral load of 7 × 104Fauver J.R. Petrone M.E. Hodcroft E.B. Shioda K. Ehrlich H.Y. Watts A.G. et al.Coast-to-coast spread of SARS-CoV-2 during the early epidemic in the United States.Cell. 2020; 181: 990-996.e5Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar copies/μL and an approximate CT of 20. Viruses were isolated from original clinical specimens at the University of Vermont Biosafety Level 3 (BSL-3) facility under an approved Institutional Biosafety protocol, as previously.42Bruce E.A. Mills M.G. Sampoleo R. Perchetti G.A. Huang M.-L. Despres H.W. Shirley D.J. Jerome K.R. Greninger A.L. Botten J.W. Predicting infectivity: comparing four PCR-based assays to detect culturable SARS-CoV-2 in clinical samples.medRxiv. 2021; ([Preprint] doi:10.1101/2021.07.14.21260544)Google Scholar Vero E6-TMPRSS2 cells [obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB number JCRB1819] were inoculated with 100 μL of clinical sample, inoculated for 1 hour at 37°C with rocking, washed with phosphate-buffered saline, and overlaid with 1 mL standard Vero medium containing 2% fetal bovine serum. Wells were monitored daily for the appearance of cytopathic effect.43Graham N.R. Whitaker A.N. Strother C.A. Miles A.K. Grier D. McElvany B.D. Bruce E.A. Poynter M.E. Pierce K.K. Kirkpatrick B.D. Stapleton R.D. An G. van den Broek-Altenburg E. Botten J.W. Crothers J.W. Diehl S.A. Kinetics and isotype assessment of antibodies targeting the spike protein receptor-binding domain of severe acute respiratory syndrome-coronavirus-2 in COVID-19 patients as a function of age, biological sex and disease severity.Clin Transl Immunol. 2020; 9: e1189Crossref PubMed Scopus (21) Google Scholar Once cytopathic effect was observed, supernatants were clarified to remove cellular debris and prepared for RNA extraction by mixing 1:1 with Qiagen Buffer AVL (Qiagen, Germantown, MD). The high viral loads in supernatants after viral isolation (CT 10 and 9.5, respectively) allowed preparation of multiple dilutions and mixtures. The conse" @default.
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