A team of US-based scientists has developed an amplicon-based sequencing method for the human monkeypox virus. The method has higher viral genome coverage and can even analyze samples with low viral DNA concentration. The study is currently available on the medRxiv* preprint server.
Background
Whole genome sequencing of pathogens plays an important vital role in identifying novel variants and monitoring their propagation during any epidemic/pandemic outbreaks. During the coronavirus disease 2019 (COVID-19) pandemic, over 13 million viral genomes isolated from 103 countries have been sequenced and made publicly available. This has significantly helped accelerate the basic and clinical research focusing on disease pathogenesis, diagnostics, prophylactics, and therapeutics.
Monkeypox is a zoonotic DNA virus initially endemic to Central and Western Africa. In 2022, several outbreaks of monkeypox infections have been noticed in non-endemic countries, including the UK and USA. Initially, the metagenomics approach was employed to sequence the monkeypox genome. However, this method is expensive and requires samples with high viral nucleic acid concentrations.
In the current study, scientists have developed and validated a primer scheme for the human monkeypox virus that can be used with amplicon-based sequencing workflows.
Development and validation of primer scheme
The scientists designed the human monkeypox primer scheme with PrimalScheme using a pre-outbreak viral genome belonging to the A.1 lineage. The primer scheme comprised 163 primer pairs with an average amplicon length of 1977 bp.
The primer scheme was initially used to sequence ten clinical samples with varied ranges of viral DNA concentrations with amplicon-based sequencing and compared with metagenomic sequencing.
The findings revealed that both sequencing approaches provide comparable genome coverage for samples with high viral DNA concentrations. However, for samples with low DNA concentrations, amplicon-based sequencing resulted in significantly increased genome coverage.
Overall, these observations indicate that amplicon-based sequencing using a primer scheme can improve monkeypox genome coverage in clinical samples with low viral DNA content.
Further validation of the primer scheme was done by collaborating with 12 public health laboratories across the USA and other countries. These laboratories employed Illumina or Oxford Nanopore Technology sequencing approaches to analyze clinical samples with various viral DNA concentrations.
The findings revealed that the monkeypox primer scheme could be used with various workflows and sequencing platforms to obtain high genome coverage for samples with high or low DNA content.
Although most of these samples were lesion swabs, a few oropharyngeal swabs collected from the throat with or without lesions were sequenced using a primer scheme. The findings revealed that oropharyngeal samples could be used as an alternative sample type to obtain near-complete viral genome sequencing.
Further validation of the protocol revealed that clinical samples with a Ct value of less than 29 should be used for sequencing. At least one million reads per sample should be generated to maximize the genome coverage. Increasing the Ct values and reducing the sequencing reads per sample might result in low genome coverage, as mentioned by the scientists.
The Ct value obtained from polymerase chain reaction (PCR) is a surrogate measure of the amount of nucleic acid in samples. The higher the Ct value; the lower the nucleic acid content.
Study significance
The study describes developing and validating an amplicon-based sequencing approach for the human monkeypox virus that can be used as a low-cost and high-throughput alternative to metagenomic sequencing.
As mentioned by the scientists, this primer scheme can be used with current SARS-CoV-2 sequencing workflows and Illumina or Oxford Nanopore Technology sequencing platforms.
Significantly, this amplicon-based sequencing approach can increase monkeypox genome coverage’s sensitivity, breadth, and depth in clinical samples with low viral DNA concentrations. This will facilitate the genomic surveillance of monkeypox outbreaks worldwide.
*Important notice
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
