Optimized SARS-CoV-2 Variant Sequencing

Samples were extracted using the Qiasymphony-DSP mini kit 200 (Qiagen, UK) with offboard lysis or manually using the Qiagen mini viral extraction kit. Samples were then tested using the CDC N1 assay to confirm the Ct values before sequencing. ARTIC protocol V2 sequencing protocol was used until June 2021, after which we switched to the V3 protocol. ARTIC version 3 primers were used for the tiling PCR until we switched to the University of Zambia (UNZA) primer set that provided better results for Delta VOC in August 2021 (data not shown) [20 (link)]. Initially two primer pools were used, however a third pool was made for primer pairs that commonly had lower depth compared to the average (details Supplementary Table 1). PCR cycling conditions were adapted to the new sequencing primers, with annealing temperature changed to 60°C. Sequencing was carried out with the Oxford Nanopore Technologies MinION sequencer. Samples that had poor coverage (<70%) with the ARTIC primer set were repeated with the UNZA primer set.