Rapid PCR-Based Nanopore Adaptive Sequencing Improves Sensitivity and Timeliness of Viral Clinical Detection and Genome Surveillance

Nanopore sequencing has been widely used for the real-time detection and surveillance of pathogens with portable MinION. Nanopore adaptive sequencing can enrich on-target sequences without additional pretreatment. In this study, the performance of adaptive sequencing was evaluated for viral genome enrichment of clinical respiratory samples. Ligation-based nanopore adaptive sequencing (LNAS) and rapid PCR-based nanopore adaptive sequencing (RPNAS) workflows were performed to assess the effects of enrichment on nasopharyngeal swab samples from human adenovirus (HAdV) outbreaks. RPNAS was further applied for the enrichment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasopharyngeal swab samples to evaluate sensitivity and timeliness. The RPNAS increased both the relative abundance (7.87-12.86-fold) and data yield (1.27-2.15-fold) of HAdV samples, whereas the LNAS increased only the relative abundance but had no obvious enrichment on the data yield. Compared with standard nanopore sequencing, RPNAS detected the SARS-CoV-2 reads from two low-abundance samples, increased the coverage of SARS-CoV-2 by 36.68-98.92%, and reduced the time to achieve the same coverage. Our study highlights the utility of RPNAS for virus enrichment directly from clinical samples, with more on-target data and a shorter sequencing time to recover viral genomes. These findings promise to improve the sensitivity and timeliness of rapid identification and genomic surveillance of infectious diseases.