A rapid, cost efficient and simple method to identify current SARS-CoV-2 variants of concern by Sanger sequencing part of the spike protein gene

Abstract

In 2020, the novel coronavirus, SARS-CoV-2, caused a pandemic, which is still raging at the time of writing this. Many countries have set up high throughput RT-qPCR based diagnostics for people with COVID-19 symptoms and for the wider population. In addition, with the use of whole genome sequencing (WGS) new lineages of SARS-CoV-2 have been identified that have been associated with increased transmissibility or altered vaccine efficacy, so-called Variants of Concern (VoC). WGS is generally too labor intensive and expensive to be applied to all positive samples from the diagnostic tests, and often has a turnaround time too long to enable VoC focused contact tracing. Here, we propose to use Sanger sequencing for the detection of common variants of concern and key mutations in early 2021, using a single set of the recognized ARTIC Network primers. The proposed setup relies entirely on materials and methods already in use in diagnostic RT-qPCR labs and on existing infrastructure from companies that have specialized in cheap and rapid turnaround Sanger sequencing. In addition, we provide an automated mutation calling software (https://github.com/kblin/covid-spike-classification). We have validated the setup on 195 SARS-CoV-2 positive samples, and we were able to profile >85% of RT-qPCR positive samples, where the last 15% largely stem from samples with low viral count. At approximately 4 {euro} per sample in material cost, with minimal hands-on time, little data handling, and a turnaround time of less than 30 hours, the setup is simple enough to be implemented in any SARS-CoV-2 RT-qPCR diagnostic lab. Our protocol provides results that can be used to focus contact-tracing efforts and it is cheap enough for the tracking and surveillance of all positive samples for emerging variants such as B.1.1.7, B.1.351 and P.1 as of January 2021.