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Monkeypox

Monkeypox virus variant surveillance

Using NextGenPCR™ library preparation for Nanopore sequencing

 


Rapid genomic surveillance of zoonotic monkeypox virus (MPXV) infections can provide valuable insights into geographic/demographic viral transmission and guide public health interventions. In current MPXV sequencing approaches, nucleotides isolated from samples are directly sequenced on a nanopore sequencer. The existing methodology is inefficient due to its low genomic coverage and sequencing depth of the fragments of interest and costly because samples cannot be multiplexed.

To aid laboratories with their MPXV genetic surveillance efforts, Molecular Biology Systems has developed an ultrafast library preparation protocol for the analysis of MPXV genomes from samples. Amplification of the 190kb MPXV genome was achieved by using PCR-based amplicon tiling of 2.5kb segments (88 amplicons total) inspired by the SARS-CoV-2 Midnight Protocol and the ARTIC network. With NextGenPCR® ultrafast PCR chemistry, the complete PCR cycling program of the protocol can be completed in under 42 minutes.

The full laboratory protocol can be viewed here  : https://www.protocols.io/

 

The Monkeypox primer panel

The composition of the oligonucleotide primer panel was developed by Martin Schou Pedersen from the Department of Clinical Microbiology at Rigshospitalet, Copenhagen University Hospital, Denmark and the NextGenPCR® MPXV library preparation protocol was tested on 40 samples in close collaboration with Matthijs Welkers and Marcel Jonges from the Dept. of Medical Microbiology & Infection Prevention at Amsterdam UMC (location AMC), The Netherlands.

Using NextGenPCR® MPXV library preparation up to 20 samples could be multiplexed in a single nanopore flowcell with greatly improved sequencing depth and coverage owing to its PCR tiling based target enrichment. This additionally results in lower costs per sample. The NextGenPCR® MPXV library preparation method was successfully employed to sequence the first known case of MPXV infection in The Netherlands.


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Strengthening global surveillance of pathogens with cheaper, robust, and validated workflows that give accurate real-time sequencing data.

Monkeypox Virus Variant Surveillance