The COVID-19 pandemic is an ongoing global challenge, with the repeated emergence of new variants that are more contagious, more virulent, drug resistant or evade vaccine-induced immunity. In response, the HiFiViral SARS-CoV-2 kit was developed as a scalable solution with increased resilience against virus mutations, designed for use on the Sequel IIe system. Unlike PCR-based amplicon methods, the HiFiViral SARS-CoV-2 kit relies on ~1,000, densely tiled Molecular Inversion Probes (MIPs) such that every genomic position is covered by ~22 probes, resulting in robust genome coverage of all circulating variants, including the mutation-dense Omicron lineage, across a broad range of Ct values.
The HiFiViral kit offers many benefits for high-throughput surveillance. Sequencing 675 bp fragments with highly accurate HiFi reads enables comprehensive variant detection, including single nucleotide variants, indels, structural variants, and identification of multi-strain samples. The kit is scalable, containing all reagents needed to enrich and barcode 384 samples, in batches ranging from 24 – 384, for sequencing in one SMRT bell library. For high throughput labs, up to 8 SMRT Cells may be loaded on an instrument with no subsequent touch points, for up to 3,072 samples per run. The enrichment assay is also simpler to execute than PCR-based assays, consisting of just 4 add-only, color-change indicated steps. Barcoding primers come pre-mixed in a 384-well, resealable plate. The simple assay design uses fewer plastics, limiting the impact of supply chain issues. In addition, methods for running the HiFiViral kit were established on a mosquito® HV Genomics pipetting robot and assessed for their performance. Finally, SMRT Link data analysis is one touch, and reports include variant calling, genome coverage, multi-strain detection, and a plate performance summary. File outputs include consensus sequences ready for database submission and reads and consensus sequences aligned to the Wuhan reference.
In this study, we demonstrate consistent recovery of >95% complete SARS-CoV-2 genomes using the commercially available HiFiViral kit at 4 different sites performing routine genomic surveillance. The evaluation runs included a broad range of sample Ct inputs across control and nasopharyngeal samples in batches up to 384. The runs also demonstrated consistent performance against Alpha, Delta, Omicron and other variant lineages, without the need for probe updates. In summary, the HiFiViral for SARS-CoV-2 kit is a cost-effective, convenient, accurate method for viral sequencing and well-suited for scalable surveillance of a rapidly evolving virus to inform public health decision making.
1PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025
2SPT Labtech, Ltd., Melbourn Science Park ,Melbourn, Hertfordshire SG8 6HB