Rapid turnaround multiplex sequencing of SARS-CoV-2: comparing tiling amplicon protocol performance

Genome sequencing is pivotal to virus surveillance, revealing the emergence and global dissemination of acquired genetic mutations. Amplicon sequencing has proven effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated frequent revision of sequencing protocols in order to maintain performance for emerging virus lineages. We compared the performance of Oxford Nanopore Technologies (ONT) Midnight and ARTIC tiling amplicon protocols using 96-plexed nanopore sequencing of 193 Delta-lineage SARS-CoV-2 clinical specimens with complete paired results and 71 S-gene target failure-selected specimens collected during the emergence of Omicron BA.1. For Delta samples, ARTIC v4 recovered [≥]90% of the genome in 82.4% (159/193) of specimens compared with 71.5% (138/193) for Midnight. ARTIC v4 was more robust in low-titre, high-Ct specimens, whereas Midnight achieved slightly greater completeness at lower Ct. In the S-gene target failure-selected cohort, revised Midnight v2 and ARTIC v4.1 primer schemes substantially improved genome completeness: [≥]99% genome recovery was achieved in 74.6% (53/71) with Midnight v2 versus 18.3% (13/71) with Midnight, and 57.7% (41/71) with ARTIC v4.1 versus 0% (0/71) with ARTIC v4. Estimated consumable costs were lower for Midnight than ARTIC ({pound}12 versus {pound}16 per sample at Q1 2022 prices), and the interval from reverse transcription to flow cell loading was shorter (~7 versus ~9.5 hours). Midnight proved a practical alternative for rapid nanopore sequencing, with lower consumable costs and shorter preparation time, although ARTIC v4 was more robust in high-Ct Delta specimens.