Use of Whole Genome Sequencing Data for a First in Silico Specificity Evaluation of the RT-qPCR Assays Used for SARS-CoV-2 Detection
Authors: Steven Van Gucht
Mathieu Gand
Arnaud Capron
Kevin Vanneste
Sigrid C J De Keersmaecker
Isabelle Thomas
Nancy H C Roosens
Philippe Herman
Journal: International Journal of Molecular Sciences (2020)
Discipline: CHEMISTRY
Keywords: COVID-19; RT-qPCR; SARS-CoV-2; WGS data; bioinformatics tool
Affiliations: Transversal activities in Applied Genomics, Sciensano, J. Wytsmanstraat 14, B-1050 Brussels, Belgium., Viral Diseases, Sciensano, J. Wytsmanstraat 14, B-1050 Brussels, Belgium., Quality of Laboratories, Sciensano, J. Wytsmanstraat 14, B-1050 Brussels, Belgium., Expertise and Service Provision, Sciensano, J. Wytsmanstraat 14, B-1050 Brussels, Belgium.

Abstract:  The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus' genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants. 

Retracted: No

PubPeer comments:
 The title of the article is misleading. There are several studies addressing the same research question, but the authors still claim to be the  FIRST . Apart from the studies they missed, they cited two published studies [[28] , [35] ] and three preprints [[36] , [37] , [38] ] that addressed the same similar research question. It is not important to be the FIRST. Your research is still worthy if you are building on previous studies to partially confirm the findings and asking new questions. , 
 #1 Thank you for reading our paper. However, there is a misunderstanding. With the first in the title, we didn t want to claim to be the first to do this kind of analysis. The first in the title refers to primary analysis (as we wrote: ...for a first in silico specificity evaluation of... ) , so you first do an in silico specificity analysis using WGS data and then you perform the actual in vitro experiments, as also elaborated in the discussion. Other studies have indeed also addressed the same similar research questions, and this we have mentioned in the introduction. We will also not be the last to this kind of analysis. It will be important to repeat it, as the virus is mutating over time (and especially once a vaccine will be available). We mentioned this in the discussion. We hope that we now have clarified this issue. ,