A universal method for the extraction, enrichment, and sequencing of mitochondrial genomes for the purposes of forensic wildlife investigations

Abstract

Wildlife forensic investigations increasingly rely on molecular species identification, yet current polymerase chain reaction (PCR)-based methodologies are constrained by the need for species-specific primer sets, high reagent costs, and inefficiencies introduced by co-extracted nuclear and bacterial DNA. This study presents a universal, PCR-independent protocol for the isolation and sequencing of mitochondrial DNA (mtDNA) from non-human mammalian whole blood samples for forensic wildlife applications. Using Solid Phase Reversible Immobilization technology in conjunction with Exonuclease V enzymatic digestion (Mseek protocol), linear nuclear DNA was selectively digested and removed from tissue extracts, yielding purified circular mitochondrial genomes suitable for downstream next-generation sequencing (NGS) without PCR amplification. Co-extracted bacterial DNA was also effectively reduced through this protocol. Isolated mtDNA extracts from four felid species -- Acinonyx jubatus (cheetah), Panthera uncia (snow leopard), Panthera leo (lion), and Panthera onca (jaguar) -- were sequenced on the Illumina NextSeq 2000 platform and successfully mapped to their respective NCBI reference genomes. Post-Mseek samples demonstrated substantially higher mitochondrial mapping percentages (2.3–25.4%) compared to pre-Mseek samples (0.05–0.1%), with analysis times reduced from hours to seconds. These results demonstrate that targeted mtDNA isolation prior to NGS significantly increases sequencing efficiency, reduces per-sample cost, and eliminates dependency on known primer sequences, advancing the feasibility of a truly universal molecular method for species identification for forensic wildlife investigations.