key: cord-1052001-saacta6w
authors: Lind, Andreas; Barlinn, Regine; Landaas, Elisabeth Toverud; Andresen, Lise Lima; Jakobsen, Kirsti; Fladeby, Cathrine; Nilsen, Mariann; Bjørnstad, Pål Marius; Sundaram, Arvind Y.M.; Ribarska, Teodora; Müller, Fredrik; Gilfillan, Gregor D.; Holberg-Petersen, Mona
title: Rapid SARS-CoV-2 variant monitoring using PCR confirmed by whole genome sequencing in a high-volume diagnostic laboratory
date: 2021-07-07
journal: J Clin Virol
DOI: 10.1016/j.jcv.2021.104906
sha: a587c37f9f387c99b977f7cc3c04940471f84f65
doc_id: 1052001
cord_uid: saacta6w

OBJECTIVES: : The emerging SARS-CoV-2 variants of concern (VoC), B.1.1.7, B.1.351 and P.1, with increased transmission and/or immune evasion, emphasise the need for broad and rapid variant monitoring. Our high-volume laboratory introduced a PCR variant assay (Variant PCR) in January 2021 based on the protocol by Vogels et al. STUDY DESIGN: : To assess whether Variant PCR could be used for rapid B.1.1.7, B.1.351 and P.1 screening, all positive SARS-CoV-2 airway samples were prospectively tested in parallel using both the Variant PCR and whole genome sequencing (WGS). RESULTS: : In total 1,642 SARS-CoV-2 positive samples from individual patients were tested within a time span of 4 weeks. For all samples with valid results from both Variant PCR and WGS, no VoC was missed by Variant PCR (totalling 399 VoC detected). Conversely, all of the samples identified as “other lineages” (i.e., “non-VoC lineages”) by the Variant PCR, were confirmed by WGS. CONCLUSIONS: : The Variant PCR based on the protocol by Vogels et al., is an effective method for rapid screening for VoC, applicable for most diagnostic laboratories within a pandemic setting. WGS is still required to confirm the identity of certain variants and for continuous surveillance of emerging VoC.

 SARS-COV-2 Variant PCR based on the protocol by Vogels et al provides fast results, demands relatively few human and data resources, and delivers high throughput.  In 1,642 SARS-CoV-2 positive samples, no Variant of Concern was missed by Variant PCR, confirmed by whole genome sequencing.  All samples identified as "non-VoC lineages" by the Variant PCR, were confirmed by whole genome sequencing.  The Variant PCR based on the protocol by Vogels et al., is a good and effective method for rapid screening for variants of concern, applicable for most diagnostic high-volume laboratories.

The discovery of a new SARS-CoV-2 variant (lineage B.1.1.7 / N501Y_V1) with increasing incidence in the United Kingdom in the autumn of 2020, highlighted the importance of rapid and comprehensive surveillance of SARS-CoV-2 variants of concern (VoC) [1] . As exemplified by the arrival of the N501Y spike mutation, which was reported to contribute to increased viral transmission [2] , rapid detection of VoC has become essential to control the These include both the N501Y and E484K spike mutations, which may be important for increased viral contagion and immune evasion [3] . The gold standard for the detection of VoC is whole genome sequencing (WGS), which is a powerful tool to map and describe the viral genome, including monitoring for current and emerging virus variants and viral genome changes with possible biological significance [4, 5] . WGS is increasingly performed in several countries and has given valuable insights into the emergence and dynamics of SARS-CoV-2 variants worldwide. However, sequencing is time and resource demanding and requires extensive data processing. It is therefore less apt for the comprehensive rapid variant analysis needed to contain VoC within a population in a pandemic setting. 

The PCR assay based on the Vogels protocol (in this paper termed "Variant PCR") was established in our high-volume laboratory at Oslo University Hospital (OUH) in January 2021. To assess whether this assay could be used for rapid B. 

The based on a standard method using magnetic beads [7, 8] . Bacteriophage MS2 RNA (Merck, Sigma-Aldrich, Darmstadt, Germany) was added before extraction as internal control [9] .

Nucleic acid was isolated from 100 µl sample and eluted in 100 µl PCR-grade water. The 

Variant PCR was performed on positive samples only, using the Vogels protocol [11] . RNA from positive samples was re-extracted using magnetic beads on Tecan Fluent 1080 as described above. Real-time RT-PCR assays in 25 µl reaction volume were performed with the QuantiNova Pathogen master mix (Qiagen, Germantown, MD, USA), using CDC-N1 ("N gene"), Yale-Spike 69/70del ("Δ69/70") and Yale-ORF1a3675-3677del ("ORF1a") primer and probe sets in accordance with the protocol. Thermal cycling was performed at 50ºC for 10 min for reverse transcription, followed by 95ºC for 2 min and 40 cycles of 95ºC for 10s and 55ºC for 30s. Results indicated by target failure were interpreted in accordance with the Vogels protocol (Table 1 ). The N gene was regarded as a positive "pan-SARS-CoV-2"

control, and samples with CT values for N1 >35 or undetected (No CT) were defined as inconclusive. Likewise, samples with Δ69/70 and/or ORF1a detected, but with a CT >35

were also defined as inconclusive. and converted to FASTQ format, and the remaining steps were performed independently for each sample. Primer sequences were trimmed from the reads using pTrimmer v1.3.3 [12] .

Reads pairs that did not have known primer sequences at the beginning of both reads were discarded. The read pairs were then subjected to quality filtering and adapter trimming using fastp v0.20.1 [13] . Reads were aligned to the Wuhan-Hu-1 reference sequence (NC_045512.2; https://www.ncbi.nlm.nih.gov/nuccore/1798174254) genome using bowtie2 v2.4.0 [14] . The reads at each genomic position were first collated using samtools v1.9 mpileup (http://www.htslib.org/doc/samtools-mpileup.html), then iVar v1.3 was used to call the consensus sequence based on the mpileup output [15] . Positions covered by fewer than ten reads were masked in the consensus sequence. At each position, the nucleotide variant with the highest frequency in the reads was used. The lineages of the samples were assigned based on the consensus sequences, using Panglin and Nextclade [16] [17] [18] [19] [20] . The analysis pipeline is available at https://github.com/nsc-norway/covid-seq/tree/v7. Table 3 . 

The rapid identification of VoC is key to control the current SARS-CoV-2 pandemic. In an infection control setting, this information is important for choosing adequate containment measures on an individual and community level. We here show that it is possible to gain this information using a rapid and simple PCR-based variant assay, capable of detecting and differentiating the B. 

Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom

Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England

Resurgence of COVID-19 in Manaus, Brazil, despite high seroprevalence

Pathogen Genomics in Public Health

Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands

PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern n

Rapid and simple method for purification of nucleic acids

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Multiplexed RT-qPCR to screen for SARS-COV-2 B.1.1.7, B.1.351, and P.1 variants of concern V.2. ProtocolsIo

pTrimmer: An efficient tool to trim primers of multiplex deep sequencing data

Gencore: an efficient tool to generate consensus reads for error suppressing and duplicate removing of NGS data

Fast gapped-read alignment with Bowtie 2

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

Nextstrain: real-time tracking of pathogen evolution

A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology

Stay-At-Home Orders Are Associated With Emergence of Novel SARS-CoV-2 Variants

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.