Abstract
The global spread of SARS-CoV-2 was significantly impacted by the emergence of Variants of Concern (VOC), with Pakistan experiencing similar trends to other countries. To gain a comprehensive understanding of the genomic epidemiology of SARS-CoV-2 strains circulating during Pakistan's third and fourth pandemic waves, we conducted the largest single sequencing effort in the country to date. Using the GridION platform (Oxford Nanopore Technologies), we performed whole genome sequencing on 1052 confirmed COVID-19 patient samples collected from multiple cities across Pakistan between March and October 2021. Our analysis revealed a clear temporal shift in variant dominance. The Alpha variant (B.1.1.7 lineage) predominated in the first half of 2021, while the Delta variant (B.1.617.2) became most prevalent in the latter half. This transition reflects global trends and provides crucial insights into the timing and dynamics of this shift within Pakistan. Mutational analysis revealed that the most frequent nucleotide mutations in Pakistani SARS-CoV-2 samples were A23403G (associated with the D614G mutation in the spike protein), C3037T, C14408T, and C241T, potentially contributing to increased disease transmission and evasion of host immune responses. The rapid evolution and spread of these circulating variants highlight the possibility of novel variants emerging with enhanced mutational fitness. The AY.108 lineage, which has been reported at relatively low frequencies globally including in Europe and North America, with Pakistan accounting for approximately 34% of global cases, suggesting potential regional evolution or specific introduction events. Our findings underscore the dynamic nature of SARS-CoV-2 and emphasize the critical importance of ongoing, large-scale genomic surveillance in Pakistan. This study demonstrates the feasibility and utility of using nanopore sequencing for comprehensive viral monitoring in Pakistan's public health context. While we utilized the higher-throughput GridION platform for centralized processing, the Oxford Nanopore technology still provided distinct advantages through its simplified library preparation protocol, flexible run capacities, and reduced computational requirements for base-calling compared to traditional high-throughput sequencers. These benefits enabled efficient processing of our large sample batches collected from distributed sites across the country, demonstrating a scalable approach for genomic surveillance that balances throughput needs with resource availability. This could inform future public health strategies, including vaccine updates and targeted interventions. Continued monitoring and adaptive strategies are essential to keep pace with the evolving viral ecology and to enhance preparedness for future outbreaks.