Specific allelic discrimination of N501Y and other SARS‐CoV‐2 mutations by ddPCR detects B.1.1.7 lineage in Washington State

Abstract Real‐time epidemiological tracking of variants of concern (VOCs) can help limit the spread of more contagious forms of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), such as those containing the N501Y mutation. Typically, genetic sequencing is required to be able to track VOCs in real‐time. However, sequencing can take time and may not be accessible in all laboratories. Genotyping by RT‐ddPCR offers an alternative to rapidly detect VOCs through discrimination of specific alleles such as N501Y, which is associated with increased transmissibility and virulence. Here we describe the first cases of the B.1.1.7 lineage of SARS‐CoV‐2 detected in Washington State by using a combination of reverse‐transcription polymerase chain reaction (RT‐PCR), RT‐ddPCR, and next‐generation sequencing. We initially screened 1035 samples positive for SARS‐CoV‐2 by our CDC‐based laboratory‐developed assay using ThermoFisher's multiplex RT‐PCR COVID‐19 assay over four weeks from late December 2020 to early January 2021. S gene target failures (SGTF) were subsequently assayed by RT‐ddPCR to confirm four mutations within the S gene associated with the B.1.1.7 lineage: a deletion at amino acid (AA) 69‐70 (ACATGT), deletion at AA 145, (TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were detected in two specimens; follow‐up sequencing revealed a total of 9 mutations in the S gene and phylogenetic clustering within the B.1.1.7 lineage. Next, we continued screening samples for SGTF detecting 23 additional B.1.1.7 variants by RT‐ddPCR and confirmed by sequencing. As VOCs become increasingly prevalent, molecular diagnostic tools like RT‐ddPCR can be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of SARS‐CoV‐2.


| BACKGROUND
The first known case of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 variant in the United States was reported in Colorado on December 29th 2020, and the next day, it was confirmed in California. 1,2 University of Washington Virology had begun surveillance a few days prior using polymerase chain reaction (PCR) to screen SARS-CoV-2 positive samples for the absence, or "dropout" of the S gene, which encodes the spike (S) protein on the surface of the viral particle. The B.1.1.7 variant is characterized by 17 mutations, eight of which occur within the S gene domain. 3,4 This region of the SARS-CoV-2 genome is of interest due to the B.1.1.7 lineage being associated with increased transmissibility, but also because the approved vaccines in the United States target the S protein. [4][5][6][7][8][9][10] Over the course of four weeks, we screened more than a thousand SARS-CoV-2 positive samples for the S gene target failure (SGTF). We selected random clinical specimens that were positive using a CDC-based laboratory-developed test (LDT) for SARS-CoV-2, and typically had a cycle threshold (C T ) under 35. [11][12][13][14][15] These SARS-CoV-2 positive samples were then amplified with the TaqPath COVID-19 assay (ThermoFisher Scientific), a multiplex reverse-transcription PCR (RT-PCR) targeting the S gene, as well as regions within the N gene and ORF1ab. 16  quencing is typically used to confirm variants of concern (VOCs). 17 However, sequencing can be a time-consuming and resource-intensive process that not all laboratories have integrated into clinical workflows.
Analysis of publicly available sequencing data by the Broad Institute revealed that it takes a median of 85 days to get from sample to publicly available sequence in the United States. 18,19 Here we describe the first detection of B. 1 F I G U R E 1 RT-PCR, RT-ddPCR, and sequencing surveillance effort for initial detection of B.1.1.7 lineage. Samples sent to UW Virology for SARS-CoV-2 molecular detection were initially screened for S gene transcript failure (SGTF) by multiplex RT-PCR as a proxy for variant detection. SARS-CoV-2 positive samples with SGTF were subsequently genotyped for specific allelic discrimination by RT-ddPCR. Both SGTF samples that genotyped as B.1.1.7 lineage by RT-ddPCR were also sequenced and clustered with N501Y.V1 clades in the United States. RT-ddPCR, droplet digital RT-PCR; RT-PCR, reverse-transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 Note: Primer and probe sets used in B.1.1.7 genotyping RT-ddPCR assay for SARS-CoV-2 variant detection. In the "Sequence" column, the bolded and underlined nucleotides reflect the region of deletion or mutation. For instance, for the probe used in S1A to detect the 69-70 deletion, the sequence ACATGT is present between the bolded and underlined thymine and cytoside nucleotides in wild-type virus. In the probe used for S1B, a thymine (uracil) nucleotide is included in place of adenosine at position 18 in the probe sequence. Notably, in S2B, although the mutation is a T to G transversion, the probe sequence is a reverse complement and so is reflected by a cytosine nucleotide at position 17 in the S2B probe sequence. Primers were included at 900 nM and probes were used at 250 nM concentrations.  (Table S1). In addition, a G-block of 490 bp was designed that includes four target amplicons. All primer and probes were synthesized by ThermoFisher and the G-block was synthesized by IDT. Primers were  Table 1; the G-block sequence with un- Two multiplex RT-ddPCR reactions per sample were performed in parallel (as outlined in Table 1  Quantification of virus copies/ml is characterized in Table 2  for droplets with S1B probe amplitude above a fluorescent threshold of 5700.

| Sequencing
We obtained quality consensus genomes for both initial B.1.1.7 samples, with more than 20,000X average coverage across the SARS-CoV-2 genome (Table S4)  Panel (Thermo Fisher Scientific) is one example, however is not multiplexed and must be run in separate parallel assays. Other assays targeted N501Y as well or used melt-curve analysis, but the ABI 7500 PCR machines we used for SGTF screening are not well-suited for melt-curve analysis, and N501Y alone is not sufficient to definitely characterize a SARS-CoV-2 lineage without sequence analysis. [50][51][52] According to GISAID, the United States has only sequenced 3 out of every 1000 positive SARS-CoV-2 samples. 53 Helix, a population genomics company, initially reported that of the positive COVID-19 tests screened, less than 1% had SGTF, however, of those SGTFs, more than one-third were confirmed B.1.1.7 lineages. 54 RT-ddPCR has the potential to prioritize specimens for sequencing, allowing more efficient allocation of strain surveillance resources.
Although our surveillance was limited, the variant positivity at the time of initial detection was approximately 1 in 500 (2 out of 1035) randomly selected SARS-CoV-2 samples. However, reports indicate that due to B.1.1.7's increased transmissibility, it is already rising in frequency and is now the dominant strain in the United States as of April 2021. 6,9,42,54 As genetic surveillance becomes increasingly relevant in efforts to track and understand new SARS-CoV-2 variants of concern in real-time, RT-ddPCR continues to cement its place in the clinical laboratory armamentarium. However, RT-ddPCR technology is still not ubiquitous in clinical laboratory settings. Increased adoption and investment in this technology can allow labs to rapidly estimate the prevalence of existing variants and perform sample screening to better allocate limited sequencing resources.

AKNOWLEDGEMENTS
All the authors are gratefully acknowledged along with the laboratories involved in the generation and deposition of sequencing data, which we obtained via the GISAID Initiative (Table S2). The authors would also like to thank Victoria Mallett Rachleff for assistance in data deposition.