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Identification of nsp1 gene as the target of SARS‐CoV‐2 real‐time RT‐PCR using nanopore whole‐genome sequencing

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused the coronavirus disease 2019 (COVID‐19) pandemic. Accurate detection of SARS‐CoV‐2 using molecular assays is critical for patient management and the control of the COVID‐19 pandemic. However, there is an increasing number of SARS‐CoV‐2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence‐independent single‐primer amplification and nanopore whole‐genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5′ end of the SARS‐CoV‐2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID‐19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real‐time RT‐PCR assay. The primers and probes are highly specific for SARS‐CoV‐2. Validation with 101 clinical specimens showed that our nsp1 RT‐PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%‐97.2%), which was similar to those of N and E gene RT‐PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%‐100%). The addition of nsp1 for multitarget detection of SARS‐CoV‐2 can avoid false‐negative results due to mutations at the primers/probes binding sites of currently available RT‐PCR assays.

detection of SARS-CoV-2 can avoid false-negative results due to mutations at the primers/probes binding sites of currently available RT-PCR assays.

| INTRODUCTION
In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) cross species barrier and became the first coronavirus to be associated with a high fatality rate in humans. 1 In 2020, SARS-CoV-2, first reported from Hubei province of China in December 2019, has become the first coronavirus to cause a pandemic. 2,3 Most patients with coronavirus disease 2019 (COVID-19) present with respiratory symptoms, while 18% exhibit gastrointestinal symptoms. 4 Radiologically, COVID-19 is characterized by multifocal and peripheral ground-glass opacities, but 2.9% of severe cases did not show any abnormalities in their lung computed tomography. 2,5 Complications include acute respiratory distress syndrome, arrhythmia, secondary bacterial infection, and multiorgan failure. [6][7][8][9] Pathologically, COVID-19 was characterized by diffuse alveolar damage, endothelialitis interstitial lymphocyte infiltrates, and multinucleated syncytial cells in the lung, and microvascular steatosis in the liver. 10,11 The importance of diagnostic testing is well demonstrated with COVID-19. Due to a shortage of diagnostic tests, a large number of undiagnosed patients with relatively mild symptoms were unknowingly spreading the virus in the community, and has led to the large outbreaks in some countries. 12 Undocumented infection has been estimated to be the source of 79% of laboratory-confirmed infections. 13 Laboratory confirmation of SARS-CoV-2 relies on accurate mo- East respiratory syndrome coronavirus (MERS-CoV). 16,17 Similar to other RNA respiratory viruses, SARS-CoV-2 can mutate quickly due to the intrinsic infidelity of viral RNA polymerase. The mean evolutionary rate of SARS-CoV-2 has been estimated to be 1.8 × 10 −3 substitutions per site per year. 18 Mutations arising at the current gene targets can lower the sensitivity of the existing assays.
Currently, three major phylogenetic types have been identified, including two subclusters of type A (ancestral type), type B (characterized by T8782C of nsp4 gene and C28144T of orf8 gene) and type C (characterized by G26144T of orf3a gene). 19 According to an analysis by GISAID, 0.02% to 0.53% of the genomes contain mutations in the last five nucleotides of the 3′ ends of the published RT-PCR primer regions. 20 Therefore, there is an urgent need to expand the number of gene targets that can be used for RT-PCR diagnosis.
The aim of this study is to identify novel targets for molecular detection of SARS-CoV-2. In our previous study, we have successfully identified highly-expressed gene regions for influenza A virus using nanopore sequencing, and have developed highly sensitive and specific novel PB2 and NS RT-PCR for detection of human and zoonotic influenza A viruses. 21 Using the same strategy, we first used nanopore sequencing to identify highly-expressed gene regions of the SARS-CoV-2 genome in clinical specimens. After identifying nonstructural protein 1 (nsp1) gene as a highly-expressed gene region, we have designed the nsp1 RT-PCR which demonstrated high sensitivity and specificity using clinical specimens.  (Table S1).

| Clinical specimens
For clinical validation, we retrieved specimens that tested positive for SARS-CoV-2 using our in-house RdRp/Hel assay reported previously. 15  Sequence-independent single-primer amplification (SISPA) was performed as described previously. 2 After sequencing, Guppy v3.4.5 was used in converting the raw signal data into FASTQ format, demultiplexing, removal of nanopore and SISPA adaptor sequences. Only reads with a minimum Q score of 7 were included for subsequent analysis. The sequencing run was qualitychecked using MinIONQC. 23 Human reads were depleted by mapping to reference human genome hg38, and unmapped reads were extracted using SAMTools. 24 The nonhuman reads were mapped to the reference genome SARS-CoV-2 isolate Wuhan-Hu-1 (NCBI GenBank: MN908947.3). BCFtools Mpileup was used in creating a variant file. 25 BCFtools call, 25 vcfutils.pl, 24 and Seqtk seq 26 were used in generating the FASTA consensus sequence. Finally, the coverage data were obtained using SAMtools. 24 Only specimens with a mean coverage of 250× were included for further analysis. The consensus sequences of five specimens from four patients (HKU-902a, HKU-903a, HKU-903b, HKU-907b, and HKU-908a) were previously deposited into NCBI GenBank (accession number: MT114412, MT114414, MT114415, MT114417, MT114418). 3 The consensus sequences of HKU-904a, HKU-905a, HKU-911a, HKU-913a, and HKU-915a have been deposited into NCBI GenBank (accession number: MT365028-MT365032). Raw reads, after excluding human reads, have been deposited into BioProject (Bioproject ID: PRJNA627286).

| Phylogenetic analysis
The phylogenetic tree of the whole SARS-CoV-2 genome was constructed using neighbour-joining method using MEGA software package version 7.0. The bootstrap values from 1000 replicates were calculated to evaluate the reliability of the phylogenetic trees. Phylogenetic types A, B, and C were defined as described previously. 19

| Analytical sensitivity and specificity
The limit of detection (LOD) was determined using serially-diluted SARS-CoV-2 virus culture isolates as described previously. 15 Table 1.

| E gene real-time RT-PCR
Real-time RT-PCR for E gene was performed according to a published protocol, except that the total reaction volume was reduced to 20 μL instead of 25 μL. 14, 29 Briefly, superscript III one-step RT-PCR system CHAN ET AL.  Table S2. B, Alignment of our novel nsp1 primers and probes with SARS-CoV-2 and other human coronaviruses in the genus Betacoronavirus, and the bat coronavirus Bat/Yunnan/RaTG13/2013. Sequences from GISAID are shown in Table S2. E, envelope; N, nucleocapsid; nsp1, nonstructural protein 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 10 minutes for reverse transcription, followed by 95°C for 3 minutes and then 45 cycles of 95°C for 15 seconds, 58°C for 30 seconds.

| N gene real-time RT-PCR
Real-time RT-PCR for N gene was performed using primers and probes as described previously. 30

| Statistical analysis
Statistical analysis was performed using PRISM 6.0 for Windows. The sensitivity of nsp1 and E gene real-time RT-PCT was compared using Fisher's exact test. To identify gene regions that were highly expressed, coverage map was visualized using integrative genomics viewer. The nsp1 gene region was highly expressed in all specimens ( Figure 3A).    (Table 3).

| DISCUSSION
In this study, we have developed a highly sensitive and specific nsp1 real-time RT-PCR for the detection of SARS-CoV-2. We first in different lineages. 31 The recombination occurs in both animal and human coronaviruses. 32,33 SARS-CoV-2 is also believed to arise due to recombination event that involves the receptor-binding motif of a closely related BatcoV RaTG13. 34 Recombination is especially important because the current real-time RT-PCR targets are mainly located in the middle or 3′ end of the genome. In contrast, nsp1 gene is located in the 5′ end, and therefore, may not be affected by recombination events occurring between the nsp1 gene and RdRp gene. Unlike other targets commonly used for diagnosis which are located in the middle or 3' end of the viral genome, nsp1 is located in the 5′ end of the genome. 35 Since recombination can occur, it is important to have a target at the 5′ end of the genome.
Nsp1 is usually not considered to be highly expressed. However, our nanopore sequencing of clinical specimens has consistently shown that nsp1 gene region is highly expressed. Traditionally, it was thought that the subgenomic sequences arise from the leader sequence.
However, recently, it was shown that the subgenomic sequences arise at the nsp1-truncated nsp2 and/or truncated nsp3 region. 36 Shortly after the announcement of SARS-CoV-2 as the causative pathogen of COVID-19, Shirato et al 30 has reported the use of a conventional nested RT-PCR targeting nsp1 gene, but require Sanger sequencing for confirmation. In this early report, the sensitivity and specificity of their conventional nsp1 gene nested RT-PCR was not reported.
The coverage map for SARS-CoV-2 from our study using of SISPA and nanopore sequencing suggests that nsp1 was highly expressed in clinical specimens. In contrast, in a previous study, nsp1 was not shown to be highly expressed in the coverage map from Illumina sequencing. 37 One possibility is the difference in library preparation. In the paper by Wu  There are several limitations of this study. First, although our nanopore SISPA-based whole-genome sequencing allowed us to identify highly-expressed gene regions, there can still be bias during the library preparation which may affect the coverage of each position in the viral genome. Second, our nanopore sequencing did not encompass phylogenetic type C strains, which is mainly found in Europe and in America.

| CONCLUSIONS
We have found that viral gene nsp1 is highly expressed in patients with COVID-19. Our novel real-time RT-PCR assay targeting nsp1 was highly sensitive and specific in the detection of SARS-CoV-2 in clinical specimens. Our real-time RT-PCR is unique in that nsp1 gene is located at the 5′ end of the SARS-CoV-2 genome, while other currently published real-time RT-PCR protocols target genes located in the middle or 3′ end of the viral genome. The incorporation of the nsp1 gene in multiplex RT-PCR assays will likely improve the detection of SARS-CoV-2.

ACKNOWLEDGMENTS
We gratefully acknowledge the originating and submitting laboratories who contributed sequences to GISAID (Supporting Information Data