Monkeypox virus whole genome sequencing using combination of NextGenPCR and Oxford Nanopore v1
Year: 2022
Type: preprint
Abstract: Rapid genomic surveillance of monkeypox virus (MPXV) can provide valuable insights in order to guide public health interventions. Current sequencing protocols make use of direct Oxford Nanopore Sequencing. However, the obtained depth is a limiting factor which prevents multiplexing samples on a flowcell making sequencing very costly. Here, we provide the protocol for a PCR-based amplicon tiling approach (inspired by SARS-CoV-2 Midnight Protocol by Nikki Freed et. al. and the ARTIC network) for MPXV consisting of a total of 88 primer sets divided over 2 amplicon pools. The amplicon size is ~2,5kB. Our approach will increase the coverage (depth) significantly and allow for multiplexing up to 20 samples on a single Nanopore flowcell. In our experience clinical samples can be successfully sequenced with CT-values <25. Homopolymer regions will remain an issue in our approach, requiring manual curation of obtained consensus sequence genomes.
Language: English
Subfield: Virology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4404876973