Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
Broňa Brejová, Kristína Boršová, Viktória Hodorová, Viktória Čabanová, Askar Gafurov, Dominika Fričová, Martina Neboháčová, Tomáš Vinař, Boris Klempa, Jozef Nosek
Abstract
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 PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.