Mpox Viral Lineage Analysis and Technique Development Using Next-generation Sequencing Approach.

Background: In response to Mpox endemic and public health emergency, DCHHS aimed to develop NGS based techniques to streamline Mpox viral clade and lineage analysis.

Methods: The Mpox sequencing workflow started with DNA extraction and adapted Illumina's COVIDSeq assay using hMpox primer pools from Yale School of Public Health. Sequencing steps included cDNA amplification, tagmentation, PCR indexing, pooling libraries, sequencing on MiSeq, data analysis, and report generation. The bioinformatic analysis comprised read assembly and consensus sequence mapping to reference genomes and variant identification, and utilized pipelines including Illumina BaseSpace, NextClade, CLC Workbench, Terra.bio for data quality control (QC) and validation.

Results: In total, 171 mpox samples were sequenced using modified COVIDSeq workflow and QC metrics were assessed for read quality, depth, and coverage. Multiple analysis pipelines identified the West African clade IIb as the only clade during peak Mpox infection from July through October 2022. Analyses also indicated lineage B.1.2 as the dominant variant comprising the majority of Mpox viral genomes (77.7%), implying its geographical distribution in the United States. Viral sequences were uploaded to GISAID EpiPox.

Conclusions: We developed NGS workflows to precisely detect and analyze mpox viral clade and lineages aiding in public health genomic surveillance.