Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus

Abstract

The 2022 multi-country monkeypox outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early human monkeypox virus infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the current outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there is an urgent need for a more sensitive and broadly applicable sequencing approach. Amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for monkeypox virus with amplicon-based and metagenomic sequencing approaches. Upon comparison, we found notably higher genome coverage across the virus genome, particularly in higher PCR cycle threshold (lower DNA titer) samples, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach. By sending out primer pool aliquots to laboratories across the United States and internationally, we validated the primer scheme in 12 public health laboratories with their established Illumina or Oxford Nanopore Technologies sequencing workflows and with different sample types across a range of Ct values. Our findings suggest that amplicon-based sequencing increases the success rate across a wider range of viral DNA concentrations, with the PCR Ct value threshold at which laboratories were able to achieve 80% genome coverage at 10X ranging between Ct 25-33. Therefore, it increases the number of samples where near-complete genomes can be generated and it provides a cost-effective and widely applicable alternative to metagenomics for continued human monkeypox virus genomic surveillance. Importantly, we show that the human monkeypox virus primer scheme can be used with currently implemented amplicon-based SARS-CoV-2 sequencing workflows, with minimal change to the protocol.

A new and efficient enrichment method for metagenomic sequencing of Monkeypox virus

“…The proposed protocol has proven to be suitable for samples with high viral load (Ct values <30 in qPCR), but not so efficient in high Ct samples. In this case, amplicon-based sequencing protocols may be more useful, as different approaches have helped to improve coverage of MPXV genomes from samples with high Ct values [15,16]. However, some ampliconbased sequencing protocols show some limitations.…”

“…DNA prep paired-end libraries (Illumina, CA, USA) were prepared using 1-5 ng of DNA extracted following the blue), and orange areas highlight the study's samples. MP01bCHUAC and MP03bCHUAC followed a non-enrichment method, the rest of samples followed the enrichment method manufacturer's protocol, except for the number of PCR cycles (15). Libraries concentration was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, MA, USA).…”

Bioinformatic approaches to draft the viral genome sequence of Canary Islands cases related to the multi-country Monkeypox virus 2022-outbreak

“…However, the costs per sample as well as the viral DNA concentration requirements in the patient samples hinders an extended use. After this work was completed [19], a more sensitive approach based on tiling amplicon sequencing both for Illumina and ONT workflows was developed and validated across a number of laboratories for MPXV [41]. Despite this method being more cost-efficient, a periodic long-read metagenomics sequencing was recommended by the authors to monitor the emergence of viral variants with genomic rearrangements.…”

Perspective on the application of genome sequencing for monkeypox virus surveillance

“…Several optional strategies in nucleic acid extraction had been developed to improve the assembly quality. Although clinical specimens with low cycle threshold (Ct) values can be directly sequenced using mNGS without special treatment ( Chen et al 2023 ), the criteria of Ct still need to be clearly defined due to the lack of experimental data.…”

“…Besides, an amplification-based technique has been developed for clinical specimens with low viral loads ( Chen et al 2023 ). It has demonstrated superior performance compared to metagenomic next-generation sequencing (mNGS) in samples with a viral cycle threshold (Ct) value exceeding 18.…”