In silico evaluation of the impact of the Omicron variant on the sensitivity of RT-qPCR assays for SARS-CoV-2 detection using whole genome sequencing

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VoC) Omicron (B.1.1.529) has rapidly spread around the world presenting a new threat to global public human health. Due to the large number of mutations possessed by Omicron, concerns have emerged over potentially reduced diagnostic accuracy of reverse transcription polymerase chain reaction (RT-qPCR), the gold standard diagnostic test for SARS-CoV-2. Here, we aimed to assess the impact of Omicron on the integrity and sensitivity of RT-qPCR assays used for coronavirus disease-2019 (COVID-19) diagnosis via in silico analysis employing whole genome sequencing data and evaluated the potential for false negatives or test failure due to mismatches between primers/probes and viral genome. and BA.2 Omicron sublineage genomes, false negative results were observed for assays ChinaCDC N, Thai N, HKUniv RdRp/Hel, SigmAldr S5, SigmAldr S6 and HKUniv S. In this study, we observed three (25%) assays (ChinaCDC N, Thai N, and HKUniv RdRp/Hel) demonstrated potential for false negatives for the SARS-CoV-2 Omicron BA.1 sublineage, while four (33.3%) assays (ChinaCDC N, Thai N, HKUniv RdRp/Hel, HKUniv S, SigmAldr S5 and SigmAldr S6) demonstrated potential false negative results for the for SARS-CoV-2 Omicron BA.2 sublineage, which also has the potential for Spike (S) gene dropout despite lacking 69-70 deletion in the S gene. Further, amplicon clustering and additional substitutions analysis along with the sensitivity analysis could be used for modication and development of RT-qPCR assays for detection of SARS-CoV-2 Omicron VoC lineages. available in databases such as National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Inuenza Data (GISAID). In this study, the in silico sensitivity of 12 RT-qPCR tests (containing 30 primers and probe sets) developed for detection of SARS-CoV-2 was assessed for use in detecting SARS-CoV-2 BA.1 and BA.2 Omicron VoC sublineages as classied by Pangolin, obtained after removing redundancy from publicly available genomes from NCBI and GISAID databases. Mismatches between the amplicon regions of the SARS-CoV-2 Omicron VoC and primers and probe sets were evaluated and the clustering analysis of the corresponding amplicon sequences was carried out. For BA.1 Omicron sublineage, assays ChinaCDC N, Thai N, and HKUniv RdRp/Hel could not produce a theoretical positive signal, whereas, for BA.2 Omicron sublineage, theoretical positive signal could not be obtained for assays ChinaCDC N, Thai N, HKUniv RdRp/Hel, HKUniv S, SigmAldr S5 and SigmAldr S6. amplicon clusters consisting of one large cluster and other smaller clusters indicating higher diversity in these regions of the SARS-CoV-2 Omicron BA.1 sublineage genome. Intriguingly, USCDC N1 and SigmAldr S6 assays sensitivity was determined to be more than 99%, despite showing the largest amplicon diversity. SARS-CoV-2 Omicron VoC BA.2 sublineage, the amplicon sequences for corresponding primers/probes could be clustered in only one cluster, this could be due to a very limited number of genome sequences available for this Omicron VoC BA.2 sublineage. genomes investigated. These substitutions along with mismatches in primers and probe sequences determined by SCREENED could be considered for development of new primers and probes sets for the RT-qPCR assays used to detect the SARS-CoV-2 Omicron VoC. HKUniv RdRp/Hel assays the criteria by SCREENED our analysis, which is also the criteria elaborated give a may result in failure or to other sets and this in agreement in the and reverse primer annealing sites respectively in all ve genomes in this The HKUniv RdRp/Hel assay a in the 3’ of the reverse primer all ve genomes. The lowest sensitivity for SigmAldr S5 and SigmAldr S6 assays mismatches,respectively, in the reverse primer annealing genomes of SARS-CoV-2 Omicron BA.2 sublineage. HKUniv S assay primer annealing SARS-CoV-2 Omicron BA.2 assays BA.2 SARS-CoV-2 SARS-CoV-2 deletion in the S protein. Many mutations in the primers and probe sets of the RT-qPCR assays were seen, resulting in low sensitivity of the assays reective of the high number of mutations in the SARS-CoV-2 Omicron VoC. Apart from the sensitivity analysis, the amplicon sequence clustering and the additional substitutions in the primer and probe sets in a large number of genomes revealed the potential new primer and probe sequences that could be used for the development of RT-qPCR tests for detecting the SARS-CoV-2 Omicron VoC sublineages. As the number of SARS-CoV-2 Omicron VoC sequences is increasing rapidly, our analysis on a larger dataset could reveal more mutations and amplicon clusters and could provide more insights on the specicity of RT-qPCR assays, particularly for SARS-CoV-2 Omicron BA.2 sublineage, as well as the newly identied SARS-CoV-2 Omicron BA.3 sublineage. The effect of mismatches in the primers and probe sets revealed in this study on the sensitivity of RT-qPCR assays could be further investigated in the wet laboratory for preparation of more specic diagnostics for SARS-CoV-2 Omicron VoC detection. Lastly, given the number of unresolved potential issues with COVID-19 diagnosis testing with respect to Omicron VoC, symptomatic patients, vulnerable patients, or those with high-risk contact with infected patients, but testing negative, should be conrmed true negative by a second assay (i.e. alternative RT-qPCR assay or lateral ow assay).


Introduction
The emergence of the newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.529 lineage presents a new threat to global public human health and coronavirus disease-2019 (COVID-19) pandemic containment efforts. 1 Due to an unprecedented number of mutations, the high potential for immune escape, and the rapid global spread and increasing number of cases, the World Health Organization (WHO) labeled B.1.1.529 as Omicron Variant of Concern (VoC) and has called for immediate global action in response. 2 SARS-CoV-2 is a positive-sense, single stranded RNA virus, with a genome of ~30,000 base pairs in length. 3 As o cially recommended by the WHO 4

and
International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) 5 the current gold standard diagnostic assay for detecting SARS-CoV-2 in human specimens is a Nucleic Acid Ampli cation Test (NAAT); more speci cally, a quantitative reverse transcription polymerase chain reaction (RT-qPCR), to detect viral RNA in clinical specimens, most commonly, from nasopharyngeal or oropharyngeal swabs. RT-qPCR employs 3 oligonucleotides: a forward primer, a reverse primer, and a probe, which speci cally hybridize with respective sequence targets in the SARS-CoV-2 genome between where the forward and reverse primers recognize. 6 Subsequent ampli cation of the viral genome and production of a uorescence indicator by the probe during repeated application cycles, leads to identi cation and possible quanti cation of SARS-CoV-2 genetic material. 6 Given the high accuracy and rapid turnaround times, NAAT are the most widely used tests by clinical laboratories for COVID-19 diagnostics. 5 However, this accuracy is threatened by the rapid evolution of SARS-CoV-2, in which some RT-qPCR assays may lose sensitivity due to mutations in the viral genome of newly evolving SARS-CoV-2 VoC. 6,7 The most likely sources of RT-qPCR false negative results are due to pre-analytical errors. 8 These errors can be in specimen collection (e.g. swab), handling, transport and storage, inappropriate and/or inadequate swab quality, and/or volume. 8 Other analytical errors, such as testing carried out outside of the proper diagnostic window, as well as mismatches between primers/probes and the viral genome due to mutations, remain a threat to diagnostic accuracy in the context of novel SARS-CoV-2 VoC. 8 Mutations located in regions hybridizing with the 3' end of primers are the most sensitive, with a single mismatch in an annealing site leading to inhibition of ampli cation and potential reduced diagnostic accuracy and/or false negative results. [9][10][11][12][13] Mutations located in regions hybridizing with 5' end of primers or other annealing segments of the oligonucleotide may have a more variable impact; however 2-3 mismatches in such regions may result in reduced RT-qPCR performance. [9][10][11][12][13] Give that Omicron displays a high mutational pro le, evaluation of all diagnostic assays (molecular and antigen immunoassays) should be urgently performed to ensure diagnostic accuracy and proper identi cation of COVID-19 cases, enabling contact tracing and quarantine procedures to limit community transmission. Indeed, the United States (U.S.) Food and Drug Administration (FDA) has already noted potential issues with several diagnostic assays in the context of the Omicron VoC. 14 With respect to RT-qPCR, previous in silico studies evaluating less mutated variants has demonstrated several mismatches, including mutations located in oligonucleotide annealing sites, which may reduce the sensitivity for some NAAT. 7,15,16 RT-qPCR primers and probe are generally designed to hybridize with relatively conserved sequences in the SARS-CoV-2 RNA genome, with most assays targeting one of more of the following: Spike (S), Envelope (E), and Nucleocapsid (N) structural proteins and Open Reading Frame (ORF) ORF1ab, which encodes the RNA-dependent RNA polymerase (RdRp). 5-7, 15,16 Mutations in all of the above proteins are observed in the Omicron VoC, including over 30 nonsynonymous mutations in the S, a nonsynonymous mutation in the E, multiple amino acid substitution and a deletion in the N, and multiple nonsynonymous mutations in ORF1ab, including the nonstructural protein (nsp)12, the RdRp, a de ned target for multiple anti-viral drugs, such as molnupiravir 17 and remdesivir. 18 An overview of the mutations found in the sublineages of Omicron VoC are provided in Table 1 E  C26270T  T9I  M  A26530G  D3G  C26577G  Q19E  G26709A  A63T  N  C28311T  P13L  28362_28370del  ERS31del  G28881A, G28882A, G28883C  RG203KR  A29510C  S413R  nucleotide  C3037T  -T5386G  C4321T  A9424G  C10198T  G10447A  C12880T  C15240T  C15714T  A20055G  C25000T  C25584T  C26858T  A27259C  C27807T  A28271T  ORF1ab  T670G  S135R  C2790T  T842I  A2832G  K856R  G4184A  G1307S  6513_6515del  SL2083I  G8393A  A2710T  C9344T  L3027F  C9534T  T3090I  C9866T  L3201F  C10029T  T3255I  C10449A  P3395H  11288_11296del*  SGF3675del*   T11296G #  F3677L #   G11287T #  L3674F #  A11537G  I3758V  C14408T  P4715L  C17410T  R5716C  A18163G  I5967V  C19955T  T6564I  ORF3a  C26060T  T223I  ORF6  G27382C, A27383T, T27384C  D61L  S  C21618T  T19I  21633_21641del  LPPA24S  C21762T  A67V  21765_21770del  HV69del  C21846T  T95I  G21987A (outlier); 21987_21995 (main)  G142D  21987_21995  VYY143del  22194_22196del  NL211I  T22200G  V213G  22205GAGCCAGAAins  215EPEins  G22578A  G339D  C22674T  S371F  T22673C, C22674T  S371L  T22679C  S373P  C22686T  S375F  A22688G  T376A  G22775A  D405N  A22786T  R408S  G22813T  K417N  T22882G  N440K  G22898A  G446S  G22992A  S477N  C22995A  T478K  A23013C  E484A  A23040G  Q493R  G23048A  G496S  A23055G  Q498R  A23063T  N501Y  T23075C  Y505H  C23202A  T547K  A23403G  D614G  C23525T  H655Y  T23599G  N679K  C23604A  P681H  C23854A  N764K  G23948T  D796Y  C24130A  N856K  A24424T  Q954H  T24469A  N969K  C24503T  L981F Notes: Blue represents mutations unique to SARS-CoV-2 Omicron BA.1 sublineage. Yellow represents mutations unique to SARS-CoV-2 Omicron BA.2 sublineage. No color represents mutations in the SARS-CoV-2 Omicron VoC. *: The exact position of this deletion is ambiguous in nucleotide coordinates. #: Position of the mutation depends on the position of the deletion.
Here, we aimed to assess the potential impact of Omicron VoC on the integrity of RT-qPCR assays currently used for COVID-19 diagnosis and evaluate the potential for false negatives or test failure due to mismatches between primers/probes and viral genome. Ideally the evaluation of diagnostic assays should be performed in vitro by quali ed personnel at clinical and research laboratories. However, given the limited access to the Omicron VoC, including the multiple sublineages, limited availability of reagents and assays due to global laboratory shortages and lack of personnel as recently noted in a survey by the American Association of Clinical Chemistry 20 and all primers/probes needed to performed such comprehensive evaluation, a bioinformatics approach, using an in silico speci city (sensitivity) evaluation, as recently demonstrated by Gand et al. 7,15 , Khan et al. 16 and Nayar et al. 6 can be a useful initial and rapid evaluation. Thus, it is critical to take full advantage of the immense efforts of the scienti c community, especially in South Africa, to generate whole genome sequencing (WGS) data that are made publicly available in databases such as National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian In uenza Data (GISAID).
In this study, the in silico sensitivity of 12 RT-qPCR tests (containing 30 primers and probe sets) developed for detection of SARS-CoV-2 was assessed for use in detecting SARS-CoV-2 BA.1 and BA.2 Omicron VoC sublineages as classi ed by Pangolin, obtained after removing redundancy from publicly available genomes from NCBI and GISAID databases. Mismatches between the amplicon regions of the SARS-CoV-2 Omicron VoC and primers and probe sets were evaluated and the clustering analysis of the corresponding amplicon sequences was carried out. For BA.1 Omicron sublineage, assays ChinaCDC N, Thai N, and HKUniv RdRp/Hel could not produce a theoretical positive signal, whereas, for BA.2 Omicron sublineage, theoretical positive signal could not be obtained for assays ChinaCDC N, Thai N, HKUniv RdRp/Hel, HKUniv S, SigmAldr S5 and SigmAldr S6.

Materials And Methods
The methodology followed in this study involves collection of whole genome sequencing data for Omicron VoC from databases and assessing the impact of RT-qPCR assays on these genomes. An overview of the methodology is presented in Figure 1.

Primers and Probes Dataset
Primers and probes used in this investigation consisted of 12 RT-qPCR tests targeting different regions of the SARS-CoV-2 genome that were obtained from a WHO report 4 and/or identi ed via literature reviews [21][22][23][24][25] and evaluated in previous studies. 6,7,15,16 A total of 30 primers and probes sets (24 primers and probe sets using TaqMan technology and 6 primer sets using SYBR Green technology) were used. These are part of 12 RT-qPCR tests developed for the detection of SARS-CoV-2 (Table 2 and Supplementary le 3). Targets of the 12 RT-qPCR tests are located in the SARS-CoV-2 genes coding for the E, N and S structural proteins, and the non-structural proteins (nsp) RdRp/helicase (Hel) and 14 (nsp14) located in the viral ORF1ab.

Selection of representative genomes by sequence identity clustering
Clustering of the Omicron VoC sequences was performed to remove redundancy in the dataset. All the downloaded sequences were clustered using CD-HIT-EST vr. 4.6.8 (https://github.com/weizhongli/cdhit) with sequence identity cut-off equal to 1.0 (other parameters were left at default settings). A total of 1,178 clusters of sequences were obtained from CD-HIT-EST for SARS-CoV-2 Omicron BA.1 sublineage and 5 clusters for BA.2 sublineage. Representative genomes of lower quality, i.e., showing more than three ambiguous nucleotides (such as "N") in the genomic regions targeted by the evaluated RT-qPCR assays were removed. 7,15 Total sequences after removing the redundancy and taking high-quality sequences were 232 for BA.1 and 5 for BA.2 sublineage of Omicron VoC.
2.4. Assessment of the RT-qPCR signals using SCREENED SCREENED (polymeraSe Chain Reaction Evaluation through largE-scale miNing of gEnomic Data) version 1.0, developed by Vanneste et al. 13 was employed to determine the theoretical production of RT-qPCR signals. For each PCR method to be evaluated, SCREENED rst searches for the amplicon in the genomes to be assessed using BLAST and then searches in these found amplicons whether the provided forward primer, reverse primer, or the probe, will successfully anneal. 13,15 Mismatches in the primer and the probe sequences with the genomic sequence were then evaluated for the production of a positive or negative RT-qPCR signal.
In this study, the following 'a priori' determined settings were used to de ne a positive RT-qPCR signal, using SCREENED: i. No mismatch was observed in the initial 5 nucleotides of primers' 3' end; ii. Total number of mismatches was not more than 10% of oligonucleotides length; and, iii. 90% or more of the oligonucleotides sequence accurately aligned with their targets. [9][10][11][12][13]15 These criteria were selected in accordance with the European Commission (EC) guidance on COVID-19 diagnostic performance criteria 26 and from scienti c literature evaluating the potential for mismatches affecting the performance of PCR-like methods and were used in previous studies. 7,13,15 In our study, only 1-2 mismatches were allowed for primers and probe sets (according to EC criteria 26 ), except for the forward primer of Assay HKUniv S, for which no more than 3 mismatches were allowed since it was 30 nucleotides in length. Advanced options such as the amplicon and fragment extension, and greedy clustering of the amplicon were enabled in SCREENED. Primers and probe sequences were aligned with their respective representative sequences from the clustered amplicons using MUSCLE (multiple sequence comparison by log-expectation). 27 Input les to the SCREENED were a fasta le of the genome sequences (232 for BA.1 and 5 for BA.2) and a tab-delimited text le containing primers and probes sequences to be evaluated and their corresponding amplicon sequence to be extracted in the genomes.

Evaluation of in silico analytical sensitivity
Sensitivity was used to evaluate the RT-qPCR tests used. We considered a theoretical RT-qPCR positive signal if the assay followed the criteria used by SCREENED. The sensitivity, known as the potential of a method to detect varying targets by a positive relation, could be referred to as inclusivity, which is used to evaluate performance of the assay. 28 The in silico sensitivity of an assay is more qualitative than quantitative as it signi es whether the genome is detected and could relate with the diagnostic sensitivity of the assay. 29 The in silico sensitivity (i.e. inclusivity) was calculated as the ratio of number of genomes detected, i.e., producing a positive RT-qPCR signal to the total number of genomes analyzed. 13 The in silico sensitivity was evaluated as shown in Equation 1.  Table 3 indicate that all of the 232 SARS-CoV-2 Omicron BA.1 sublineage genomes produced a false negative (genome could not be ampli ed in silico by the RT-qPCR assay) result for HKUniv RdRp/Hel assay because of a substitution in the rst ve nucleotide of the reverse primer's 3' end (C to T) resulting in 0% sensitivity of this test. A sensitivity of 1.29% was observed for ChinaCDC N assay owing to the substitution in the forward primer's 5' end (3nucleotide substitution, GGG to AAC) and resulted in false negative result for 229/232 genomes. For Thai N assay, 226/232 genomes could not be detected correctly, which resulted in a 2.59% sensitivity because of 10 nucleotide mismatches in the reverse primer. A 99.57% sensitivity was obtained for assays CoremCharite E, Pasteur E, RoujianLu ORF1a, SigmAldr N2, SigmAldr S5, SigmAldr S6, and Huang E. All other assays showed 100% sensitivity as presented in Table 3, which means that these assays resulted in in silico ampli cation of all SARS-CoV-2 Omicron BA.1 sublineage genomes included in our analysis.  "-" represents that no mismatches are obtained by SCREENED.

( )
*: False negative results represents that the primer/probe could not amplify the target in silico.

For SARS-CoV-2 Omicron BA.2 sublineage
A total of only 5 genomes of SARS-CoV-2 Omicron BA.2 sublineage were analyzed for the in silico evaluation of the RT-qPCR assays; results are presented in Table 4. Assays ChinaCDC N and Thai N showed a sensitivity of 0%, as all of the 5 genomic sequences produced false negative results. A 0% sensitivity was obtained for assay HKUniv RdRp/Hel because of a substitution in the rst ve nucleotides of the reverse primer's 3' end (C to T). Assays SigmAldr S5 and SigmAldr S6 showed a sensitivity of 20% as these could not correctly detect four out of ve sequences and had mismatches in the reverse primers. Assay HKUniv S showed 12 mismatches between the reverse primer sequence and the annealing sequence in the amplicon for three genomes, resulted in a 40% sensitivity. All other assays showed 100% sensitivity and could correctly amplify in silico all SARS-CoV-2 Omicron BA.2 sublineage genomes (Table 4).  "-" represents that no mismatches are obtained by SCREENED.
*: False negative results represents that the primer/probe could not amplify the target in silico.

Analysis of the amplicon clusters and additional substitutions in the primer and probe sequences
SCREENED also clustered the amplicon sequences from all genomes analyzed in our study targeted by the evaluated primers/probes. For each RT-qPCR assay, genomes were clustered based on the identical amplicon sequence, where the greater the number of clusters represents a higher diversity of the genomic region. The total number of clusters obtained for each assay and the redistribution of genomes in the top three clusters (from largest to smallest) are shown in Table 5. The largest cluster of the corresponding amplicon sequences for Pasteur RdRp IP2, USCDC N1, HKUniv N, HKUniv S, and SigmAldr S6 assays, targeting RdRp, N and S genes, was present in 90 to 97% of the genomes but for most of the assays, above 97% of the amplicon sequences could be clustered in one large cluster. The USCDC N1 assay with seven amplicon clusters and SigmAldr S6 assay with six amplicon clusters showed higher number of amplicon clusters consisting of one large cluster and other smaller clusters indicating higher diversity in these regions of the SARS-CoV-2 Omicron BA.1 sublineage genome. Intriguingly, USCDC N1 and SigmAldr S6 assays sensitivity was determined to be more than 99%, despite showing the largest amplicon diversity. For SARS-CoV-2 Omicron VoC BA.2 sublineage, the amplicon sequences for corresponding primers/probes could be clustered in only one cluster, this could be due to a very limited number of genome sequences available for this Omicron VoC BA.2 sublineage. Sequence alignment of the amplicons present in the largest cluster with the corresponding primer/probe sequences were performed to explore the additional substitutions or mismatches present in majority of amplicons of the genomes, apart from the ones that prevent the in silico ampli cation by the primers/probes obtained by SCREENED. This approach was also adopted by Gand et al. 7,15 Additional substitutions were present in the sequences of almost all the RT-qPCR assays (Table 6) except in ChinaCDC ORF1ab, CoremCharite N, Pasteur RdRp IP2, Pasteur RdRp IP4, HKUniv S, Won S, SigmAldr N1, and Huang E assays. These substitutions were observed in the rst and the largest clusters of the respective primers and probe sets, which contained >90% of the genomes investigated. These substitutions along with mismatches in primers and probe sequences determined by SCREENED could be considered for development of new primers and probes sets for the RT-qPCR assays used to detect the SARS-CoV-2 Omicron VoC.

Discussion
With the increasing number of mutations in the SARS-CoV-2 emerging VoC, as in B.1.1.529, also known as Omicron, the evaluation of the current RT-qPCR assays used for the detection of SARS-CoV-2 is important for correct diagnosis. Evaluation of these assays in the wet laboratory is limited in this rapidly evolving Omicron outbreak, because of the time constraint and lack of representative strains available for clinical laboratories, as previously noted. 30 Therefore, an in silico approach was used to evaluate the sensitivity of current RT-qPCR assays using the whole genome sequencing data of the SARS-CoV-2 Omicron VoC (particularly from the publicly available GISAID database), and employing suitable bioinformatics tools. 7, 30 We evaluated the sensitivity of 30 RT-qPCR primers and probe sets in this study using SCREENED, which produces alignment statistics and thus, number of false-negative results could be obtained. An overall summary of the results is presented in Table 7.  In comparison with previous studies evaluating the sensitivity of the RT-qPCR assays for SARS-CoV-2 genomes from April 2020 to January 2021, 7,15 ChinaCDC N assay had the lowest sensitivity, which is in line with our observations in this study with Omicron VoC. Further, we observed a sensitivity of 2.59% for the Thai N assay and 0% for the HKUniv RdRp/Hel assay for the Omicron VoC, while Gand et al. 15 for the same assays obtained a sensitivity of 99.73% and 100%, respectively, analyzing other VoC, which points towards important differences between the Omicron VoC and previous SARS-CoV-2 variants.
Interestingly, CoremCharite N and Pasteur RdRp IP4 assays showed the best results in our study, as these showed the highest sensitivity (Table 7), less diversity of amplicon among the genomes (Table 7), and no substitutions in the primers and probes annealing site. The Pasteur RdRp IP4 assay was intended to be speci c to SARS-CoV-2, however, the speci city of assay CoremCharite N was not communicated. 15 For SARS-CoV-2 Omicron BA.2 sublineage, which was restricted to ve genomes due to very limited number of genomes available at time of this study, ChinaCDC N, Thai N, HKUniv RdRp/Hel assays displayed the lowest sensitivity. ChinaCDC N and Thai N assays had 3 and 10 mismatches in the forward and reverse primer annealing sites respectively in all ve genomes analyzed in this study. The HKUniv RdRp/Hel assay showed a substitution in the 3' end of the reverse primer for all ve genomes. The second lowest sensitivity was observed for SigmAldr S5 and SigmAldr S6 assays with 13 and 12 mismatches,respectively, in the reverse primer annealing site for four genomes of SARS-CoV-2 Omicron BA.2 sublineage. The HKUniv S assay showed a 40% sensitivity with 12 mismatches in the reverse primer annealing site for three SARS-CoV-2 Omicron BA.2 sublineage genomes. Hence, ChinaCDC N, Thai N, HKUniv RdRp/Hel, HKUniv S, SigmAldr S5 and SigmAldr S6 assays do not meet the criteria for an appropriate primers and probe set for detecting BA.2 sublineage of the SARS-CoV-2 Omicron VoC. For SARS-CoV-2 Omicron BA.2 sublineage, other primers and probe sets ) sensitivity was in agreement with the a priori criteria.
It is likely that SARS-CoV-2 Omicron BA.2 sublineage, due to lacking the deletion in S at residues 69-70, is not "identi ed" as likely Omicron due to no S gene target dropout on some NAAT, which is often a pretext to sequencing, thus leading to underrepresentation of this sublineage in WGS data. However, in our study with the primers and probes employed, the S gene dropout could not be explained clearly for the SARS-CoV-2 Omicron BA.1 results as HKUniv S, Won S, SigmAldr S5 and SigmAldr S6 assays have high sensitivity (Table 7), which implies that these assays could correctly detect most of the studied SARS-CoV-2 Omicron BA.1 genomes included in our analysis. Thus, care should be taken in validating the use of the S gene target dropout for each speci c RT-qPCR assay as means for tracking the SARS-CoV-2 Omicron BA.1 sublineage. Interestingly, for SARS-CoV-2 Omicron BA.2 genomes, the S gene dropout could be potentially observed in HKUniv S, Won S, SigmAldr S5 and SigmAldr S6 assays, which we observed to have low sensitivity (Table 6). Conversely, the presence of the N gene dropout for SARS-CoV-2 Omicron BA.1 could potentially be observed for some assays, such as ChinaCDC N and Thai N. The presence of the N gene and/or S gene dropout on RT-qPCR are not likely to occur for the SARS-CoV-2 Delta VoC, and if detected, should alert to a high likelihood of being the Omicron VoC. 14 However, the potential for both N and S genes dropout highlights the importance of using an assay with multiple gene targets and the need for additional testing approaches in cases of suspected false negatives.
This preliminary study was limited by a small number of currently available SARS-CoV-2 Omicron VoC genomes, as well as by the rapid genetic diversi cation of the B.1.1.529, with multiple sublineage, including a newly classi ed SARS-CoV-2 Omicron BA.3. 31 However, our ndings demonstrate the urgent need to evaluate NAAT for detection of SARS-CoV-2 Omicron and other potentially similar emerging VoC. We will continue to update our analysis in the weeks ahead.
Importantly, the large number of RT-qPCR tests currently being performed employ commercially developed primers and probes, whose sequences are unknown and often not shared due to concerns over intellectual property. However, in these unprecedented times, false negative results can have detrimental consequences, especially early in the SARS-CoV-2 Omicron outbreak, limiting capacity for viral tracing, intervention, and interrupting the transmission chain.
Thus, wet laboratory evaluation and validation of commercial assay primer and probes should be urgently performed. We amplify the call by Metzger et al. 30 for governmental bodies or neutral institutions to be tasked with in silico evaluation in emergency settings for global spread of a new SARS-CoV-2 VoC to inform clinical research laboratories of potential diagnostic performance issues while protecting intellectual property.

Conclusion
In our in silico analysis evaluating 12 RT-qPCR assays with a total of 30 primers and probes, for the SARS-CoV-2 Omicron BA.1 sublineage, three (25%) assays (ChinaCDC N, Thai N, and HKUniv RdRp/Hel) demonstrated potential for false negatives, while for SARS-CoV-2 Omicron BA. in the primers and probe sets revealed in this study on the sensitivity of RT-qPCR assays could be further investigated in the wet laboratory for preparation of more speci c diagnostics for SARS-CoV-2 Omicron VoC detection. Lastly, given the number of unresolved potential issues with COVID-19 diagnosis testing with respect to Omicron VoC, symptomatic patients, vulnerable patients, or those with high-risk contact with infected patients, but testing negative, should be con rmed true negative by a second assay (i.e. alternative RT-qPCR assay or lateral ow assay).

Declarations Con icts of Interests: None
Funding: None