Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays

ABSTRACT Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ CT ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

Abstract 23 SARS-CoV-2 variants of concern (VOC) pose an increased risk to public health due to higher transmissibility 24 and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 25 mutation panel consisting of ten RT-PCR genotyping assays compared to whole genome sequencing (WGS) for 26 identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N=664), collected during 27 routine PCR screening (15≤Ct≤32) between May-July 2021 and December 2021-January 2022, were randomly 28 selected and analyzed using ten RT-PCR genotyping assays. VOC lineage was determined based on the 29 detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research 30 panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha 31 (N=207); 48.9% as Delta (N=325); 19.4% as Omicron (N=129), 0.3% as Beta (N=2) and one sample as a non-32 VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable 33 accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable and the costs and 34 turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-35 2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for 36 identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in 37 SARS-CoV-2 surveillance testing. 38 Key words: SARS-CoV-2; Variant of Concern; RT-PCR genotyping assay 39 40 Importance 41 The SARS-CoV-2 genome changes constantly. It is estimated that there are thousands of variants of  CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due 43 to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists and 44 public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, 45 and develop counter measures like vaccines, and is therefore very important. The technique used for the 46 pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of 47 standard for identification of new viral variants, it is resource-intensive, not easily scalable, requires expensive 80 equipment that is not available in most labs, and time-to-result can delay a timely public health response. 81 In the current study, we evaluated the accuracy and sensitivity of a custom TaqMan SARS-CoV-2 82 mutation panel consisting of ten RT-PCR genotyping assays classifying MOCs by comparing the results to 83 WGS for SARS-CoV-2 VOC detection in COVID-19 patients. The study was initiated between May and July 84 2021 when Delta replaced Alpha as the dominant variant in the Netherlands. Subsequently, the detection of 85 MOCs was also evaluated during December 2021 and January 2022, to monitor the emergence of Omicron in 86 The Netherlands. 87 88

Clinical nasopharyngeal samples and SARS-CoV-2 detection 90
Nasopharyngeal swabs were collected daily from May to July 2021 and from December 2021 to January 2022 91 at the public health service centers in four Dutch provinces -Flevoland, Gelderland, Limburg and Noord-92 Brabant. Upon sample collection, the swabs were placed into tubes containing 2 mL of lysis/binding buffer 93 (Roche Diagnostic, Mannheim, Germany). The tubes were transported to the National Screening Laboratory of 94 Sanquin (NSS) in Amsterdam and in this laboratory, the samples were tested for the presence of SARS-CoV-2 95 RNA using the cobas SARS-CoV-2 PCR test on the cobas 8800 System (Roche Diagnostics,Mannheim,96 Germany). The cobas SARS-CoV-2 PCR test amplifies two targets in ORF1a/b and E-gene within the SARS-97 CoV-2 genome and contains internal control RNA molecules to check for nucleic extraction and amplification 98 efficiency. 99 From all cobas SARS-CoV-2 PCR positive tested samples, 696 were randomly selected for inclusion in 100 the study: N=664 with 15≤Ct≤32 for testing the accuracy of the RT-PCR genotyping assays, and N=32 with Ct-101 values ranging from 32≤Ct≤39 to test the sensitivity of these genotyping assays. 102 To validate the RT-PCR genotyping assays (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 103 a panel of SARS-CoV-2 positive samples (N=13) was provided by The National Institute for Public Health and 104 Environment (RIVM), covering the VOC Alpha, Beta, Gamma, Delta and Omicron, former VOI Epsilon, Eta, Iota 105 and Zeta, and including the lineages B.1.177 (a major mostly European lineage, February 2020) and B.1.258.21 106 (The Netherlands lineage, October 2020) (https://cov-lineages.org/lineage_list.html). 107 The samples used in this study were obtained from individuals who visited public health service centres 108 that were part of the national SARS-CoV-2 screening and surveillance system organised by the RIVM. Upon 109 testing, visitors were informed that, if tested SARS-CoV-2 positive, their samples could be used for virus typing 110 to study the spread of SARS-CoV-2 within the Netherlands without further consent. Samples were anonymised 111 prior to investigation. All procedures were carried out in accordance with the ethical standards of the Helsinki 112 Declaration and the Netherlands Code of Conduct for Research Integrity. 113

RNA extraction 114
Regarding samples collected from May to July 2021, total RNA was extracted using the EasyMag total nucleic 115 acid extractor (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. Briefly, 350 µL 116 of the nasopharyngeal sample diluted in lysis/binding buffer was added to EasyMag vessels containing 2 mL 117 bioMerieux lysis buffer. After addition of 50 µL silica, the samples were incubated at room temperature for 10 118 minutes. Next, samples were processed according to the specific B2.0.1. protocol. The elution volume was 55 119 µl. Nucleic acid extracts were stored at <-80°C until further processing. 120 Samples collected between December 2021 and January 2022 underwent RNA extraction using the 121 QIAsymphony DSP Virus/Pathogen kit on a QiaSymphony instrument (all Qiagen, Hilden, Germany) according 122 to the manufacturer's instructions. Samples were processed using the Complex400_V4_DSP protocol. 400 µL 123 of lysate was used and the elution volume was 85 µL. Nucleic acid extracts were stored at <-80°C until further 124 processing. 125

Whole genome sequencing 126
Whole genome SARS-CoV-2 sequencing was performed using the Ion AmpliSeq SARS-CoV-2 research panel 127 on an Ion S5 system (Thermo Fisher Scientific, Waltham, Massachusetts, USA)  in the phylogenetic analysis due to multiple regions with inconclusive sequencing (a high number of N's). After 142 creating an alignment in Mega v. 11.0.8, a maximum likelihood (ML) approach with a general time reversed 143 (GTR) model was used to construct the phylogeny. Bootstrap values (N=100) were calculated to analyze the 144 stability of the tree topology. 145

RT-PCR genotyping assays 146
Ten RT-PCR genotyping assays of the TaqMan SARS-CoV-2 mutation panel (Thermo Fisher Scientific,147 Waltham, Massachusetts, USA) were selected as shown in Table 1. Assays were performed in a reaction 148 volume of 10 µL using a QuantStudio 5 Real-time PCR instrument (QS5) (Applied Biosystems, CA, USA) 149 following the manufacturer's protocol. For each sample, 5 µL of isolated SARS-CoV-2 RNA sample was added 150 to 5 µL of TaqPath 1-step RT-PCR Master Mix containing two sequence-specific unlabeled primers and two 151 allele-specific TaqMan probes. One TaqMan probe is specific for the wild type sequence and is VIC labeled, 152 while the other probe is mutant-specific and labeled with FAM dye on the 5´ end. The genotyping assays were 153 run with the QS5 instrument using the following protocol: 50˚C for 10 min, 95˚C for 2 min, 45 cycles at 95˚C for 154 3 s and 60˚C for 30 s. Post-readings were carried out at 50˚C. The data were analyzed using Design and 155 Analysis Software 2.5.0 and the VOC lineage was determined based on the detected mutation profile (Table 1). 156

Results 158
Validation of the RT-PCR genotyping assays using reference material 159 To validate the accuracy of selected RT-PCR genotyping assays of the TaqMan SARS-CoV-2 mutation panel, 160 we tested a panel of reference samples (N=13) provided by the RIVM containing SARS-CoV-2 positive samples 161 with known whole genome sequences and therefore known types of selected SARS-CoV-2 variants: Alpha, 162 Beta, Gamma, Delta, Epsilon, Eta, Iota, Omicron, Zeta, as well as lineages B.1.258.21 and B.1.177. All samples 163 were tested using selected genotyping assays and the results showed a 100% concordance (Supplementary 164 Table S3). 165

CoV-2 VOC in comparison to WGS 167
Taking into account the VOCs circulating at the time, we selected nine RT-PCR genotyping assays to test the 168 331 randomly selected SARS-CoV-2 positive samples collected in the period of May-July 2021 in two regions of 169 the Netherlands -Flevoland and Limburg ( Figure 1A). Compared with the mutation profile, the RT-PCR 170 genotyping assays classified 207/331 (62.5%) of the samples as Alpha; 121/331 (36.6%) as Delta and 2/331 171 (0.6%) as Beta SARS-CoV-2 variants. One sample was classified as a non-VOC based on our selected RT-172 PCR genotyping assays. In 331 out of 331 samples, matching results were obtained using WGS (100%). The 173 sample which could not be assigned to a specific lineage using RT-PCR genotyping assays was determined by 174 WGS as the C.36.3.1 variant. Our data demonstrates the accuracy of the RT-PCR genotyping assays in VOC 175 detection. Furthermore, our dataset showed a rapid increase in Delta variant prevalence from 0% for the first 176 week of June 2021 to 100% of SARS-CoV-2 cases by mid July 2021 ( Figure 1B). 177

Tracking the increase in prevalence of the Omicron SARS-CoV-2 variant using RT-PCR genotyping 178 assays in comparison to WGS 179
After the first reports of Omicron SARS-CoV-2 variant, we included a novel RT-PCR genotyping assay for 180 Omicron-specific mutation Q493R in our mutation panel to detect the Omicron SARS-CoV-2 positive samples, 181 both subtypes BA.1 and BA.2, and differentiate them from the Delta variant which still accounted for almost all 182 cases detected at the beginning of December 2021. Based on the VOCs circulating at the time, we selected 183 three RT-PCR genotyping assays ( Figure 1C) and tested 333 SARS-CoV-2 positive samples collected between and Noord-Brabant. Based on the mutation profile, the RT-PCR genotyping assays classified 204/333 (61.3%) 186 as Delta and 129/333 (38.7%) as Omicron SARS-CoV-2 variants, with two samples belonging to the BA.2 187 sublineage of Omicron. In 333 out of 333 samples, matching results were obtained using WGS (100%). These 188 results demonstrated the accuracy of the RT-PCR genotyping assays in VOC detection. Furthermore, our data 189 showed a shift of the Omicron variant prevalence from 0% to 100% in a time frame of seven weeks ( Figure 1D).

Sensitivity of the RT-PCR genotyping assays versus WGS in SARS-CoV-2 samples with low viral loads 197
To compare the sensitivity between both surveillance methods, RT-PCR genotyping assays and WGS were 198 performed on 32 SARS-CoV-2 positive samples with high Ct-values ranging from 32 to 39 cycles. The  genotyping assays were able to successfully classify SARS-CoV-2 VOCs in 30 out of 32 samples (93.8%), 200 while WGS established the VOCs in 29 out of 32 samples (90.6%). One out of 32 samples failed both 201 approaches (3.1%), whereas three samples failed one out of two methods (WGS:RT-PCR genotyping assays 202 2:1) (9.4%). However, the obtained results of those three samples were of poor quality, resulting in many and 203 large regions with inconclusive sequencing for WGS and the lack of a genotyping call for the Delta-specific RT-204 PCR genotyping assay (L452R  (20), are widely used and the value of these assays has previously been shown. During the 211 ongoing COVID-19 pandemic, genomic surveillance efforts have been performed worldwide and constitute an 212 extremely valuable tool for monitoring SARS-CoV-2 evolution and its associated disease severity, as well as the 213 performance of vaccines, therapeutics, diagnostic tools, or other public health measures. Although WGS is a 214 critical tool for identification of new emerging variants, there is a need to develop faster, accurate, and cost-215 effective methods for SARS-CoV-2 variant surveillance, which enable quick public health responses and are 216 also applicable in lower resource settings. 217 This study evaluated the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 218 ten RT-PCR genotyping assays detecting MOC based on allelic discrimination for SARS-CoV-2 VOC detection 219 in comparison to WGS. The concordance between the two methods was 100%, including one sample classified 220 as a non-VOC based on our selected RT-PCR genotyping assays which was determined by WGS as the 221 C.36.3.1 variant, which demonstrated the accuracy of genotyping assays for VOC surveillance. Moreover, RT-222 PCR genotyping assays were able to identify the shift of the Delta variant prevalence from 0% to 100% in a 223 timeframe of eight weeks (May-July 2021) and a shift of the Omicron variant prevalence from 0% to 100% 224 between December 2021 and January 2022 in the Netherlands which further emphasized the relevance of 225 genotyping assays for epidemiological surveillance. 226 Although our study provided SARS-CoV-2 VOC classification in a highly accurate manner, several 227 limitations apply. First, genotyping assays rely on pre-existing knowledge of the SARS-CoV-2 genomic 228 sequences. Given the rapid emergence of new mutations and therefore new variants which can contain multiple 229 co-occurring mutations, the design and selection of probe sets for genotyping assays will always depend on 230

WGS. 231
While WGS is advocated by the WHO for the identification and confirmation of SARS-CoV-2 variants, 232 this approach has several limitations as well. WGS has higher cost, longer turnaround times and the complexity 233 of implementation in laboratories compared with RT-PCR genotyping assays. Thus, RT-PCR genotyping 234 assays could constitute an alternative to WGS that allows a rapid turnaround time (<8 hours versus 3-4 days by 235 WGS), is easy to implement and cost-effective. The cost for the mutation based approach even with a panel of 236 ten RT-PCR genotyping assays would be 15 times cheaper compared to WGS with possibility of greater 237 savings if using a smaller panel. Once new mutations have been classified by WGS, custom RT-PCR 238 genotyping assays could be set up within two weeks. 239 Another technique that could be considered for the routine detection of emerging SARS-CoV-2 VOC is 240 a Multiplex PCR using melting curve analysis. Multiplex PCR is a useful method for the detection of SARS-CoV-241 2 VOC that produces reliable results, but requires a nested PCR step which can increase the risk of 242 contamination. In addition, allele-specific probes are used to perform multiplex PCR, that is why melting curves 243 must be performed for confirmation.(21) Furthermore, this technique may increase the risk of unclear or 244 erroneous measurement due to the additional genetic variation in target regions which would require additional 245 training of laboratory employees to interpret complex genetic information about a rapidly mutating virus. (22, 23)  246 As a result, genotyping assays could be easier to implement in laboratories than any currently available 247 alternative methods. 248 Although the analytical sensitivity of the RT-PCR genotyping assays has not been measured in this 249 study, Peterson et al. (2022) used a similar method to classify SARS-CoV-2 genomic variants in wastewater 250 and they showed that the approach exhibited a significant sensitivity with the limits of detection (LOD) ranging 251 from 3 to 6 copies/reaction.(24) Similar studies using one or more genotyping assays to detect mutations in the 252 S-and/or ORF8-gene reported the successful use of this approach for detection of SARS-CoV-2 VOC.(25-27) 253 Therefore, RT-PCR genotyping assays should be considered as an accurate and sensitive approach that could 254 be executed in any standard laboratory. 255 Although PCR-based genotyping approaches will not replace WGS as the reference standard for the 256 SARS-CoV-2 surveillance testing, they constitute an easily implementable and scalable tool complementary to 257 WGS. Furthermore, the costs and turnaround time of genotyping approach are significantly reduced, and a new 258 genotyping assay can be set up within two weeks when a new mutation is discovered by WGS. Finally, since 259 evolution of viral mutation patterns in a country or region may indicate the emergence of new SARS-CoV-2 260 variants, conducting mutation surveillance using genotyping assays might be useful to identify a signal to start 261   Table 1: The ten SARS-CoV-2 RT-PCR genotyping assays, detecting mutations of concern based on allelic 383 discrimination, of a custom TaqMan SARS-CoV-2 mutation panel which have been used in this study. show a result which can look as a heterozygous call and can therefore signal the presence of a different mutation at 387 that position, /: the delY144 assay was designed to detect the coresponding mutation in the Alpha variant of concern 388 and could not be applied for the Omicron variant due to additional mutations being present in the adjectent positions 389 that would compromise the ability of probes and primers to bind appropriately. Telephone: (0031) 20 512 1347 20 21 SARS-CoV-2 variants of concern (VOC) pose an increased risk to public health due to higher transmissibility 23 and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 24 mutation panel consisting of ten RT-PCR genotyping assays compared to whole genome sequencing ( The SARS-CoV-2 genome changes constantly. It is estimated that there are thousands of variants of SARS-41 CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due 42 to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists and 43 public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, 44 and develop counter measures like vaccines, and is therefore very important. The technique used for the 45 pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of 46 SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building 47 blocks is presented. This method enables a fast, accurate, and cheap determination of different SARS-CoV-2 48 VOC and would be a powerful method to include in SARS-CoV-2 surveillance testing. The presence of overlapping mutations of concern (MOC) in different VOCs was used to develop the 75 described RT-PCR-based approach for variant surveillance. As each VOC carries MOCs in different 76 combinations along with additional mutations, genotyping of a few specific genomic positions enables rapid 77 detection and discrimination between different VOCs. While whole genome sequencing (WGS) is the reference 78 standard for the identification of new viral variants, it is resource-intensive, not easily scalable, requires 79 expensive equipment that is not available in most labs, and time-to-result can delay a timely public health 80 response. 81 In the current study, we evaluated the accuracy and sensitivity of a custom TaqMan  The samples used in this study were obtained from individuals who visited public health service centers 108 that were part of the national SARS-CoV-2 screening and surveillance system organized by the RIVM. Upon 109 testing, visitors were informed that, if tested SARS-CoV-2 positive, their samples could be used for virus typing 110 to study the spread of SARS-CoV-2 within the Netherlands without further consent. Samples were anonymized 111 prior to the investigation. All procedures were carried out under the ethical standards of the Helsinki Declaration 112 and the Netherlands Code of Conduct for Research Integrity. 113

RNA extraction 114
acid extractor (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. Briefly, 350 µL 116 of the nasopharyngeal sample diluted in lysis/binding buffer was added to EasyMag vessels containing 2 mL 117 bioMerieux lysis buffer. After the addition of 50 µL silica, the samples were incubated at room temperature for 118 10 minutes. Next, samples were processed according to the specific B2.0.1. protocol. The elution volume was 119 55 µl. Nucleic acid extracts were stored at <-80°C until further processing. 120 Samples collected between December 2021 and January 2022 underwent RNA extraction using the 121 QIAsymphony DSP Virus/Pathogen kit on a QiaSymphony instrument (all Qiagen, Hilden, Germany) according 122 to the manufacturer's instructions. Samples were processed using the Complex400_V4_DSP protocol. 400 µL 123 of lysate was used and the elution volume was 85 µL. Nucleic acid extracts were stored at <-80°C until further 124 processing. 125  Table S1) (https://www.epicov.org/epi3/frontend#483d2c). 138 A phylogenetic tree was constructed containing (near) full genome sequences (N=660; 15≤Ct≤32) plus 139 nine reference sequences from GenBank matching the nine SARS-CoV-2 clades of the (near) full genome 140 sequences (Supplementary Table S2). Four sequences of B.1.1.7 (N=1) and BA.1 (N=3) had not been included 141 in the phylogenetic analysis due to multiple regions with inconclusive sequencing (a high number of N's). After 142 creating an alignment in Mega v. 11.0.8, a maximum likelihood (ML) approach with a general time-reversed 143 (GTR) model was used to construct the phylogeny. Bootstrap values (N=100) were calculated to analyze the 144 stability of the tree topology. 145

RT-PCR genotyping assays 146
Ten RT-PCR genotyping assays of the TaqMan SARS-CoV-2 mutation panel (Thermo Fisher Scientific,147 Waltham, Massachusetts, USA) were selected as shown in Table 1. Assays were performed in a reaction 148 volume of 10 µL using a QuantStudio 5 Real-time PCR instrument (QS5) (Applied Biosystems, CA, USA) 149 following the manufacturer's protocol. For each sample, 5 µL of isolated SARS-CoV-2 RNA sample was added 150 to 5 µL of TaqPath 1-step RT-PCR Master Mix containing two sequence-specific unlabeled primers and two 151 allele-specific TaqMan probes. One TaqMan probe is specific for the wild-type sequence and is VIC labeled, 152 while the other probe is mutant-specific and labeled with FAM dye on the 5´ end. The genotyping assays were 153 run with the QS5 instrument using the following protocol: 50˚C for 10 min, 95˚C for 2 min, 45 cycles at 95˚C for 154 3 s and 60˚C for 30 s. Post-readings were carried out at 50˚C. The data were analyzed using Design and 155 Analysis Software 2.5.0 and the VOC lineage was determined based on the detected mutation profile (Table 1). 156

Results 158
Validation of the RT-PCR genotyping assays using reference material 159 To validate the accuracy of selected RT-PCR genotyping assays of the TaqMan SARS-CoV-2 mutation panel, 160 we tested a panel of reference samples (N=13) provided by the RIVM containing SARS-CoV-2 positive samples 161 with known whole genome sequences and therefore known types of selected SARS-CoV-2 variants: Alpha, 162 Beta, Gamma, Delta, Epsilon, Eta, Iota, Omicron, Zeta, as well as lineages B.1.258.21 and B.1.177. All samples 163 were tested using selected genotyping assays and the results showed 100% concordance (Supplementary  164   Table S3). 165

CoV-2 VOC in comparison to WGS 167
Taking into account the VOCs circulating at the time, we selected nine RT-PCR genotyping assays to test the 168 331 randomly selected SARS-CoV-2 positive samples collected in the period of May-July 2021 in two regions of 169 the Netherlands -Flevoland and Limburg ( Figure 1A). Compared with the mutation profile, the RT-PCR 170 genotyping assays classified 207/331 (62.5%) of the samples as Alpha; 121/331 (36.6%) as Delta and 2/331 171 (0.6%) as Beta SARS-CoV-2 variants. One sample was classified as a non-VOC based on our selected RT-172 PCR genotyping assays. In 331 out of 331 samples, matching results were obtained using WGS (100%). The 173 sample which could not be assigned to a specific lineage using RT-PCR genotyping assays was determined by 174 WGS as the C.36.3.1 variant. Our data demonstrate the accuracy of the RT-PCR genotyping assays in VOC 175 detection. Furthermore, our dataset showed a rapid increase in Delta variant prevalence from 0% for the first 176 week of June 2021 to 100% of SARS-CoV-2 cases by mid-July 2021 ( Figure 1B). RT-PCR assays for the genotyping of viruses, such as hepatitis viruses (17) (20), are widely used and the value of these assays has previously been shown. During the 211 ongoing COVID-19 pandemic, genomic surveillance efforts have been performed worldwide and constitute an 212 extremely valuable tool for monitoring SARS-CoV-2 evolution and its associated disease severity, as well as the 213 performance of vaccines, therapeutics, diagnostic tools, or other public health measures. Although WGS is a 214 critical tool for the identification of new emerging variants, there is a need to develop faster, more accurate, and 215 cost-effective methods for SARS-CoV-2 variant surveillance, which enable quick public health responses and 216 are also applicable in lower resource settings. 217 This study evaluated the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 218 ten RT-PCR genotyping assays detecting MOC based on allelic discrimination for SARS-CoV-2 VOC detection 219 in comparison to WGS. The concordance between the two methods was 100%, including one sample classified 220 as a non-VOC based on our selected RT-PCR genotyping assays which was determined by WGS as the 221 C.36.3.1 variant, which demonstrated the accuracy of genotyping assays for VOC surveillance. Moreover, RT-222 PCR genotyping assays were able to identify the shift of the Delta variant prevalence from 0% to 100% in a 223 timeframe of eight weeks (May-July 2021) and a shift of the Omicron variant prevalence from 0% to 100% 224 between December 2021 and January 2022 in the Netherlands which further emphasized the relevance of 225 genotyping assays for epidemiological surveillance. 226 Although our study provided SARS-CoV-2 VOC classification in a highly accurate manner, several 227 limitations apply. First, genotyping assays rely on pre-existing knowledge of the SARS-CoV-2 genomic 228 sequences. Given the rapid emergence of new mutations and therefore new variants which can contain multiple 229 co-occurring mutations, the design, and selection of probe sets for genotyping assays will always depend on 230

WGS. 231
While WGS is advocated by the WHO for the identification and confirmation of SARS-CoV-2 variants, 232 this approach has several limitations as well. WGS has a higher costs, longer turnaround times, and the 233 complexity of implementation in laboratories compared with RT-PCR genotyping assays. Thus, RT-PCR 234 genotyping assays could constitute an alternative to WGS that allows a rapid turnaround time (<8 hours versus 235 3-4 days by WGS), is easy to implement, and is cost-effective. The cost for the mutation-based approach even 236 with a panel of ten RT-PCR genotyping assays would be 15 times cheaper compared to WGS with the 237 possibility of greater savings if using a smaller panel. Once new mutations have been classified by WGS, 238 custom RT-PCR genotyping assays could be set up within two weeks. 239 Another technique that could be considered for the routine detection of emerging SARS-CoV-2 VOC is 240 a Multiplex PCR using melting curve analysis. Multiplex PCR is a useful method for the detection of SARS-CoV-241 2 VOC that produces reliable results, but requires a nested PCR step which can increase the risk of 242 contamination. In addition, allele-specific probes are used to perform multiplex PCR, which is why melting 243 curves must be performed for confirmation. (21) Furthermore, this technique may increase the risk of unclear or 244 erroneous measurement due to the additional genetic variation in target regions which would require additional 245 training of laboratory employees to interpret complex genetic information about a rapidly mutating virus. (22, 23)  246 As a result, genotyping assays could be easier to implement in laboratories than any currently available 247 alternative methods. 248 Although the analytical sensitivity of the RT-PCR genotyping assays has not been measured in this 249 study, Peterson et al. (2022) used a similar method to classify SARS-CoV-2 genomic variants in wastewater 250 and they showed that the approach exhibited a significant sensitivity with the limits of detection (LOD) ranging 251 from 3 to 6 copies/reaction. (24) Similar studies using one or more genotyping assays to detect mutations in the 252 S-and/or ORF8-gene reported the successful use of this approach for the detection of SARS-CoV-2 VOC. (25-253 27) Therefore, RT-PCR genotyping assays should be considered an accurate and sensitive approach that could 254 be executed in any standard laboratory. 255 Although PCR-based genotyping approaches will not replace WGS as the reference standard for the 256 SARS-CoV-2 surveillance testing, they constitute an easily implementable and scalable tool complementary to 257 WGS. Furthermore, the costs and turnaround time of genotyping approach are significantly reduced, and a new 258 genotyping assay can be set up within two weeks when a new mutation is discovered by WGS. Finally, since 259 the evolution of viral mutation patterns in a country or region may indicate the emergence of new SARS-CoV-2 260 variants, conducting mutation surveillance using genotyping assays might be useful to identify a signal to start 261 large-scale WGS promptly.  Table 1: The ten SARS-CoV-2 RT-PCR genotyping assays, detecting mutations of concern based on allelic 383 discrimination, of a custom TaqMan SARS-CoV-2 mutation panel which have been used in this study. With mut: mutation, wt: wildtype, *: the presence of another mutation at the same position would cause the assay to 386 show a result which can look as a heterozygous call and can therefore signal the presence of a different mutation at 387 that position, /: the delY144 assay was designed to detect the coresponding mutation in the Alpha variant of concern 388 and could not be applied for the Omicron variant due to additional mutations being present in the adjectent positions 389 that would compromise the ability of probes and primers to bind appropriately. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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