Monkeypox virus multiplexed PCR amplicon sequencing (PrimalSeq) v2
Authors: F.G. Chen* Nicholas 
Published: October 2022

Version 3 Updates: Updated Monkeypox virus (MPXV) nomenclature Resolved typo in step 7.3 Included recommendations for sample selection and assigning library reads Included link to validation study pre-print Complete validation study: https://doi.org/10.1101/2022.10.14.22280783 Background: The current global outbreak of human Monkeypox virus (hMPXV) concurrent with an ongoing SARS-CoV-2 pandemic has further highlighted the need for genomic surveillance and pathogen whole genome sequencing. While metagenomic and hybrid capture sequencing approaches were used to sequence many of the early hMPXV cases, the viability of these methods is dependent on 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 a strong 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 design a primer scheme for hMPXV and we validated it with widely used SARS-CoV-2 sequencing protocols. Based on initial validation, our approach shows notably higher depth and breadth of coverage across the genome, particularly with higher PCR cycle threshold (Ct) samples, as compared to metagenomic sequencing. While further testing is needed, the early success of this approach has significant implications for sequencing efforts of the current hMPXV outbreak and serves as a proof of concept of amplicon-based sequencing for use with other large-genome DNA viruses and potentially bacterial genomes. Overview of Design: We used PrimalScheme (https://primalscheme.com/) to generate an hMPXV primer scheme using a pre-outbreak A.1 lineage reference genome (GenBank accession: MT903345). The primer scheme comprises a total of 163 primer pairs with an amplicon length ranging between 1597 and 2497 bp (average length of 1977 bp). The primer scheme is compatible with current ARTIC and COVIDSeq SARS-CoV-2 sequencing protocols, and while validated only with Illumina library prep methods and sequencing platforms, it would likely see a high degree of success with other sequencing platforms such as the Oxford Nanopore Technologies MinION. Initial Validation: We validated our hMPXV primer scheme with clinical specimens at the Massachusetts Department of Public Health, Massachusetts State Public Health Laboratory under the IRB Project Titled “Rash Illness: Alternate Specimen Types and Sequencing” (protocol number 1917413). A total of 25 clinical specimens were included in this initial validation comprised of both throat and swabs of fluid from lesions of 8 individuals, belonging to the current outbreak-associated hMPXV B.1 lineage as determined by prior characterization at the Centers of Disease Control and Prevention. Ct values were determined with the non-variola orthopox diagnostic qPCR assay developed for use by the Laboratory Response Network (Rapid Diagnostic Testing for Response to the Monkeypox Outbreak — Laboratory Response Network, United States, May 17–June 30, 2022 | MMWR (cdc.gov)). Clinical samples ranged in cycle threshold (Ct) values from 15.03 (high viral concentration) to 34.63 (low viral concentration), and each sample was sequenced in parallel using a metagenomics approach and the provided amplicon-based approach. Extractions and sequencing analysis were performed in accordance with current biosafety guidance including extraction in a BSL-3 setting. An evaluation of site-specific biosafety practices should be developed in consultation with your organization’s biosafety officer. Libraries were prepared with the Illumina DNA prep kit and sequenced on the MiSeq (v2 kit running 2x150 nt reads). Consensus genomes were generated at 10X coverage using the TheiaCoV_Illumina_PE Workflow Series on Terra.bio. An hMPXV “fork” was developed for us by Curtis Kapsak from Theiagen, which included the hMPXV reference sequence, primer scheme, and consensus genome length, and can currently be accessed at: https://github.com/theiagen/public_health_viral_genomics/tree/cjk-MPXV-theiacov. We found comparable genome coverage between amplicon and metagenomic sequencing with low Ct (<18) samples, yet a significant increase in genome coverage with amplicon sequencing in higher Ct samples (>18; Figure 1A.) The library clean up stage of the amplicon based Illumina DNA prep protocol was conducted using the ‘standard DNA input’ option for comparison to metagenomics. Following optimization with 15 additional paired samples unrelated to the initial 10 samples, we found improved genome coverage with higher Ct samples (>25) using the ‘small PCR amplicon input’ option of the Illumina DNA prep protocol (Figure 1B.) Our findings highlight how amplicon-based approaches can significantly expand hMPXV sequencing to a wider variety of samples. Amongst samples with a low Ct (<18), genome coverage via amplicon sequencing was consistently >97%, with minimal amplicon drop-outs (Figure 2). Amplicons 11, 75, and 118 showed consistent drop-out across the sequenced samples, while none of the primers had mismatches, except for a single nucleotide mismatch in the 11_RIGHT primer. We did obtain coverage for these amplicons when sequencing clade IIa DNA from cultured virus (strain USA-2003; NR-4928) obtained from BEI Resources (NIAID, NIH). This suggests that the dropouts of these primers may be a specific issue related to the current hMPXV B.1 lineage genomes. As this protocol is still in development, we will further investigate performance as we continue to sequence additional samples. Conclusion: We developed an amplicon-based sequencing (PrimalSeq) approach for hMPXV that improved the depth and breadth of genome coverage with low viral concentration specimens as compared to metagenomic sequencing. This protocol represents the third iteration of development. Further versions will be uploaded to protocols.io with an accompanying description of changes as appropriate.