Validation of the ARTIC SARS-CoV-2 sequencing protocol

The SARS-CoV-2 ARTIC amplicon protocol is the most widely used genome sequencing method for SARS-CoV-2, accounting for over 43% of publicly available genome sequences. Understanding the limits of this protocol helps to inform how the data is used and interpreted. An analysis was undertaken with quantified SARS-CoV-2 control material to establish the limit of detection of the ARTIC protocol.Our data demonstrates that amplicon sequencing through the ARTIC protocol can assist diagnostics tests and is sensitive in the detection of low virion counts of SARS-CoV-2. The limit of detection for the ARTIC protocol is a minimum of 25-50 virus particles per mL. ARTIC has a comparable sensitivity to the published sensitivities of commercial assays. A set of over 2,000 clinical samples from three UK regions were then analysed to understand the limits of the ARTIC protocol on real-world samples and to understand the relative performance of clinical diagnostic assays. Positives detected by diagnostic platforms were supported by sequencing data, with platforms that used RT-qPCR being the best predictor that the sample would sequence successfully. To maximise success of sample sequencing for phylogenetic analysis, samples should be less than Ct 31. For diagnostic tests that do not provide a quantifiable value, we recommend an explicit quantification step be added to the sequencing protocol.