Comparative Study of Platform-Specific and Hybrid Assembly Approaches in SARS-CoV-2 Genome Sequencing. null

During the COVID-19 pandemic, next-generation sequencing (NGS) has been instrumental for public health laboratories in identifying and characterizing SARS-CoV-2 mutations. These genomic insights have informed national responses, from tracking viral spread to associating mutations with phenotypic traits, such as virulence and immune escape. The Illumina systems and Oxford Nanopore Technologies (ONT) have been used for NGS, each presenting unique advantages. Hybrid assembly, a bioinformatic technique often applied in characterizing new bacterial species, leverages both technologies’ strengths to produce a more accurate and higher-quality genome. While hybrid assembly has been used to enhance SARS-CoV-2 genome quality, its optimal conditions for SARS-CoV-2 sequencing and surveillance have not been systematically studied. This study fills this gap by evaluating the conditions under which hybrid assembly improves SARS-CoV-2 genomic surveillance through a comparative analysis via eight bioinformatic methods of 192 routine surveillance samples sequenced by Illumina and ONT. Our findings indicate that hybrid assembly does not significantly outperform other methods in unique mutation detection. However, it excels in generating more complete genomes, albeit marginal compared to Illumina-based methods, and reliably identifies mutations across the frequencies amongst the different methodologies. Moreover, our research underlines the critical need for in-house validation of methods and exposes the limitations inherent in proprietary pipelines. Our findings suggest hybrid assembly as a potent quality control tool in genomic surveillance, particularly beneficial for augmenting ONT-based sequencing when coupled with Illumina data. A decision tree analysis pinpointed the percentage of trimmed ONT reads as a critical predictor for successful hybrid assemblies, reinforcing the importance of high-quality ONT reads for effective hybrid assembly and underscoring its supportive role in ONT experiments. Our comprehensive evaluation of sequencing strategies provides valuable insights for public health laboratories, guiding the optimization of genomic surveillance for SARS-CoV-2 and enhancing the global response to the pandemic.