Nanopore Sequencing of SARS-CoV-2: Comparison of Short and Long PCR-tiling Amplicon Protocols

Brejova, B.; Borsova, K.; Hodorova, V.; Cabanova, V.; Gafurov, A.; Fricova, D.; Nebohacova, M.; Vinar, T.; Klempa, B.; Nosek, J.

Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with this approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, as well as the reads derived from the viral sub-genomic RNAs and/or human and bacterial contamination.