key: cord-0313625-h42nzhai authors: Nazario-Toole, A. E.; Nguyen, H. M.; Xia, H.; Frankel, D. N.; Kieffer, J. W.; Gibbons, T. F. title: Sequencing SARS-CoV-2 from Antigen Tests date: 2021-07-18 journal: nan DOI: 10.1101/2021.07.14.21260291 sha: 951a49c62a66d0d4e7920b5442ac04f7d6282606 doc_id: 313625 cord_uid: h42nzhai Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media. Early in the pandemic Mercatelli and Giorgi predicted a low mutation rate for SARS-CoV-2 based 42 on whole-genome sequencing of 48,635 specimens [1] . Continued genomic surveillance has 43 revealed a worrying mutation rate of 3.7x10 -6 per nucleotide per cycle [2] . Furthermore, the rate 44 of mutation varies across the SARS-CoV-2 genome, with several sites exhibiting recurrent 45 mutations that, due to strong positive selection, emerged independently a minimum of three times 46 in strains sequenced across the globe [3] . Ongoing genomic surveillance of SARS-CoV-2 is 47 Variants 50 The CDC defines a variant as a viral genome that has one or more mutations that differentiate it 51 from other variants in circulation [8] . Variants may have different potential impacts on public 52 health. Variants of interest (VOIs) require monitoring. Variants of concern (VOCs) may affect 53 treatment, transmission, or disease severity. Variants of high consequence (VOHC) have 54 significantly reduced effectiveness of prevention measures or therapeutics relative to existing or 55 previous variants. Fortunately, no VOHCs have been identified to date. The following VOCs 56 Although exceedingly rare, reinfection has been documented and is a threat to vulnerable 63 populations. Although most suspected cases of reinfection were a resurgence of the same viral 64 strain that initially infected the patient, other cases demonstrate reinfection with genetically distinct 65 genomes [7, 13] . Genome surveillance enables us to monitor for cases of reinfection and discern 66 whether these cases are associated with particular variants. 67 Initially, SARS-CoV-2 tests relied on nasopharyngeal swabs placed in viral transport media (VTM) 69 followed by RT To continue whole-genome sequences at institutions that have adopted antigen testing, 86 communication with clinicians is required to minimize pre-analytical factors that may lead to 87 sample degradation or discarding of samples. Institutions that choose to adopt antigen-based 88 whole genome sequence pipelines must work with clinicians and institutional review boards 89 (IRBs) to ensure that clinical specimens are retained for genome surveillance purposes. Clinical 90 researchers must also work with clinicians to ensure proper storage of antigen cards prior to 91 transportation to the sequencing laboratory. 92 Here, we tested various collection and storage parameters to optimize sample preparation for 93 SARS-CoV-2 whole genome sequencing from Ag-cards. We demonstrate that it is possible to 94 produce high quality genomic surveillance data from antigen-derived AN swabs. Specifically, we 95 validated PCR-based whole genome sequencing from AN swabs from the BinaxNOW™ COVID- This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260291 doi: medRxiv preprint This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260291 doi: medRxiv preprint to or greater than 1 are sequenced. (I) An in-house bioinformatics pipeline is used to process 124 sequence data. 125 To optimize the sample collection and preparation methods, we utilized a previously sequenced 127 SARS-CoV-2 positive NP specimen with an N1 CT of 12.28 (#5195) (Fig 2) . First, we examined 128 the potential for sample loss and degradation due to (1) exposure to the BinaxNOW™ proprietary 129 extraction buffer and (2) storage conditions (Fig 2A) . In this, and every following experiment, we 130 This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260291 doi: medRxiv preprint (20 or more reads mapped per nucleotide), and viral PANGO lineages are shown in Figure 2B . 149 As expected, the positive control VR-1986D was assigned to PANGO lineage A and all dipped 150 Ag-card specimens were assigned to PANGO lineage B.1, the same lineage assigned to NP 151 specimen 5195. IGV snapshots show that all samples map at greater than 99.5% at 20X coverage 152 across the genome (Fig 2C) . Together these finding indicated that sample exposure to Ag-card 153 buffer and testing conditions do not impact the quality of RNA and subsequent sequencing steps. into DNA/RNA shield, stored at 4°C for 48 hours, extracted, then amplified using RT-PCR (Fig 170 3A) . We hypothesized that due to the nature of the lateral flow assay, viral RNA would be 171 This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260291 doi: medRxiv preprint on average, 1.57 CTs lower than the line (± 1.16 CTs) (p-value = 0.0116). Interestingly, host RNA 174 levels were significantly higher in the swabs: RP was detected in swab specimens 4.43 cycles 175 sooner than the line (± 1.30 CTs) (p-value = 0.0001). Thus, relative to host RNA, viral RNA is 176 enriched on the positive line. As we were able to detect comparable levels of viral RNA on swabs 177 and positive lines, we next prepared libraries from three samples with N1 CT < 25 from both the 178 swab and positive line (Fig 3B and 3C) . Regardless of the sample source, sequenced specimens 179 had 20x genome coverage (20 or more reads per nucleotide) of greater than or equal to 99%, This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260291 doi: medRxiv preprint and point-of-care testing sites adopting antigen-based SARS-CoV-2 tests, fewer specimens may be available to support genomic surveillance of viral variants worldwide. To 199 facilitate future genomic surveillance efforts, we developed a workflow for SARS-CoV-2 whole-200 genome sequencing from antigen-based test specimens (Fig 1) . We were able to recover viral 201 RNA of sufficient quantity and quality for targeted sequencing of the SARS-CoV-2 genome (Fig 202 2) . We found that the quality of genome sequences derived from Ag-test samples is comparable 203 to RNA isolated from NP swabs collected for RT-PCR (Fig 2) . Furthermore, a comparison of 204 sample sources, antigen card swab vs. lateral flow assay (LFA) positive line, showed that viral 205 RNA from both sources can generate high quality sequencing libraries (Fig 3) . Here we only attempted to sequence from the BinaxNOW™ antigen test, but other antigen tests 220 may also yield viable specimens for whole-genome sequencing. This work is a first step for future 221 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260291 doi: medRxiv preprint The following primers were utilized: N1 Forward, 5'-GACCCCAAAATCAGCGAAAT-3', N1 245 This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260291 doi: medRxiv preprint Transmission dynamics and evolutionary 283 history of 2019-nCoV Mutation rate of SARS-285 CoV-2 and emergence of mutators during experimental evolution No evidence for increased 288 transmissibility from recurrent mutations in SARS-CoV-2 Effectiveness of the BNT162b2 Covid-19 Vaccine 314 against the B.1.1.7 and B.1.351 Variants CoV2373 Covid-19 Vaccine against the B.1.351 Variant COVID-19 reinfection: a rapid systematic review 320 of case reports and case series Center for Devices and Radiological Health, FDA Diagnostic Tests for SARS-CoV-2 Potential preanalytical and analytical vulnerabilities in the 326 laboratory diagnosis of coronavirus disease 2019 (COVID-19) Monitoring and Response Among U.S. Air Force Basic Military Trainees -Texas Office of the Commissioner, FDA. Coronavirus (COVID-19) Update: FDA Authorizes First 333 announcements/coronavirus-covid-19-update-fda-authorizes-first-antigen-test-help-rapid-336 detection-virus-causes Centers for Disease Control and Prevention. Interim Guidance for Antigen Testing for Joint Base San Antonio. MEDCoE Adds Antigen Testing Lab to ASSURED-SQVM diagnostics for COVID-19: addressing the why, when, 344 where, who, what and how of testing Emerging point-of-care biosensors for rapid 347 diagnosis of COVID-19: current progress, challenges, and future prospects Next generation 350 sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities Genomic sequencing of SARS-CoV-2: a guide to 353 implementation for maximum impact on public health. WHO BioFire COVID-19 Test: Instructions for Use under an 356 Emergency Use Authorization (EUA) Only The authors would like to thank the laboratories who provided the excess clinical specimens 279 used in this research. 280 281