Mpox Viral Lineage Analysis and Technique Development Using Next Generation Sequencing Approach

Abstract

Abstract Objective To develop next generation sequencing (NGS) techniques for Mpox viral clade and lineage analysis. Methods Mpox DNA was extracted from DCHHS patients using QIAamp DSP DNA Blood Mini Kit. The Mpox Sequencing workflow adapted Illumina’s COVIDSeq assay using hMpox primer pools from Yale School of Public Health. Sequencing steps started with amplifying cDNA amplicons, tagmentation, PCR indexing, pooling libraries, sequencing on Illumina MiSeq, analysis and report generation. The bioinformatic analysis comprised of read assembly and consensus sequence mapping to reference genomes and variant identification, and utilized pipelines including Illumina BaseSpace, NextClade, CLC Workbench, Terra.bio TheiaCov_Illumina_PE Workflow for data QC and validation. Results 171 Mpox samples were sequenced using modified COVIDSeq workflow and QC metrics were assessed for sequencing 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 dominant variant comprising majority of Mpox viral genomes (77.7%) implying its geographical distribution in the USA. Viral sequences were uploaded to GISAID EpiPox global database. Conclusions We developed NGS workflows to precisely detect and analyze Mpox viral clade and lineages and aid in genomic surveillance.