The integration of pathogen whole-genome sequencing with public health surveillance has proven to be a powerful tool in outbreak control. The ongoing COVID-19 pandemic has highlighted the importance of real-time genomic surveillance, leading to significant advancements in viral genomics. In the midst of this, the global outbreak of human monkeypox virus has provided a new target for genomic surveillance efforts.
In a recent publication in PLOS Biology, a group of researchers developed an amplicon-based sequencing approach for human monkeypox virus, which offers a more sensitive, cost-effective, and scalable solution compared to traditional metagenomic sequencing methods.
Understanding Human Monkeypox Virus
Human monkeypox virus is a zoonotic DNA virus endemic to Western and Central Africa. It consists of three major clades, with one subgroup (clade IIb) referred to as human monkeypox virus due to direct human-to-human transmission. The virus, initially rare outside endemic countries, has emerged as a global threat, with cases reported in non-endemic regions. The lack of consistent clinical presentations and unknown transmission dynamics necessitate the establishment of rapid genomic surveillance networks.
The Limitations of Metagenomic Sequencing
Early sequencing of human monkeypox virus genomes during the global outbreak primarily relied on metagenomics, which involves sequencing the total nucleic acid present in a sample. While effective, metagenomic sequencing comes with high resource costs, computational demands, and the requirement for high viral concentrations. These characteristics limit its suitability for rapid response in large-scale pathogen outbreaks.
Amplicon-Based Sequencing: A Game Changer
Amplicon-based sequencing, specifically the highly multiplexed PrimalSeq approach, offers a promising alternative to metagenomics. Originally developed for the Zika virus and later employed during the SARS-CoV-2 pandemic, this approach provides greater coverage, sensitivity, and throughput. By adapting PrimalSeq for human monkeypox virus, the research team achieved consistently high genome coverage across a range of PCR cycle threshold (Ct) values, workflows, and sequencing platforms.
Key Recommendations for Amplicon-Based Sequencing of Human Monkeypox Virus
Based on their findings, the researchers provided crucial recommendations for the successful implementation of amplicon-based sequencing in human monkeypox virus surveillance:
Implementation and Benefits
The primer scheme developed for human monkeypox virus can be seamlessly integrated into existing SARS-CoV-2 sequencing workflows and platforms such as Illumina and Oxford Nanopore Technologies. This streamlined approach enables public health laboratories currently using amplicon-based sequencing for SARS-CoV-2 to expand their capabilities to include human monkeypox virus surveillance without significant modifications to their workflows. Collaboration with 10 public health labs across the world confirmed the successful implementation of this approach across diverse settings, experience levels, and resource demands.
Advantages and Future Directions
Compared to metagenomic sequencing, amplicon-based sequencing demonstrated higher sensitivity, enabling the sequencing of various sample types across a wider range of Ct values. The approach also revealed limited amplicon drop-outs, indicating reliable coverage across the majority of the genome. The slower evolutionary rate of human monkeypox virus reduces the need for frequent updates to the primer scheme, simplifying its implementation. While primarily designed for clade IIb viruses, additional primer schemes may be required for other virus clades found in endemic regions.
In a recent publication in PLOS Biology, a group of researchers developed an amplicon-based sequencing approach for human monkeypox virus, which offers a more sensitive, cost-effective, and scalable solution compared to traditional metagenomic sequencing methods.
Understanding Human Monkeypox Virus
Human monkeypox virus is a zoonotic DNA virus endemic to Western and Central Africa. It consists of three major clades, with one subgroup (clade IIb) referred to as human monkeypox virus due to direct human-to-human transmission. The virus, initially rare outside endemic countries, has emerged as a global threat, with cases reported in non-endemic regions. The lack of consistent clinical presentations and unknown transmission dynamics necessitate the establishment of rapid genomic surveillance networks.
The Limitations of Metagenomic Sequencing
Early sequencing of human monkeypox virus genomes during the global outbreak primarily relied on metagenomics, which involves sequencing the total nucleic acid present in a sample. While effective, metagenomic sequencing comes with high resource costs, computational demands, and the requirement for high viral concentrations. These characteristics limit its suitability for rapid response in large-scale pathogen outbreaks.
Amplicon-Based Sequencing: A Game Changer
Amplicon-based sequencing, specifically the highly multiplexed PrimalSeq approach, offers a promising alternative to metagenomics. Originally developed for the Zika virus and later employed during the SARS-CoV-2 pandemic, this approach provides greater coverage, sensitivity, and throughput. By adapting PrimalSeq for human monkeypox virus, the research team achieved consistently high genome coverage across a range of PCR cycle threshold (Ct) values, workflows, and sequencing platforms.
Key Recommendations for Amplicon-Based Sequencing of Human Monkeypox Virus
Based on their findings, the researchers provided crucial recommendations for the successful implementation of amplicon-based sequencing in human monkeypox virus surveillance:
- Utilize existing amplicon-based SARS-CoV-2 sequencing infrastructure.
- Prioritize samples with a Ct value below 31, particularly if resources are limited.
- Generate at least 1 million sequencing reads per sample, if possible.
Implementation and Benefits
The primer scheme developed for human monkeypox virus can be seamlessly integrated into existing SARS-CoV-2 sequencing workflows and platforms such as Illumina and Oxford Nanopore Technologies. This streamlined approach enables public health laboratories currently using amplicon-based sequencing for SARS-CoV-2 to expand their capabilities to include human monkeypox virus surveillance without significant modifications to their workflows. Collaboration with 10 public health labs across the world confirmed the successful implementation of this approach across diverse settings, experience levels, and resource demands.
Advantages and Future Directions
Compared to metagenomic sequencing, amplicon-based sequencing demonstrated higher sensitivity, enabling the sequencing of various sample types across a wider range of Ct values. The approach also revealed limited amplicon drop-outs, indicating reliable coverage across the majority of the genome. The slower evolutionary rate of human monkeypox virus reduces the need for frequent updates to the primer scheme, simplifying its implementation. While primarily designed for clade IIb viruses, additional primer schemes may be required for other virus clades found in endemic regions.