{"id":{"repo_id":"central-ok","oai_identifier":"oai:shareok.org:11244/342581"},"canonical_url":"https://search.dev.ndltd.org/etd/central-ok/oai:shareok.org:11244/342581","repository":{"repo_id":"central-ok","name":"University of Central Oklahoma","base_url":"https://shareok.org/server/oai/request"},"display":{"title":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Deen, Beth A."],"institution":"Jackson College of Graduate Studies","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Creecy, James"],"committee_chairs":[],"committee_members":["Haynie, Michelle L., 1975-","Porterfield, Caitlin"],"year":2026,"date_issued":"2026","date_published":"2026","updated_at":"2026-08-21T16:43:16Z","subjects":[],"languages":[],"rights":["All rights reserved by the author, who has granted UCO Chambers Library the non-exclusive right to share this material in its online repositories. Contact UCO Chambers Library's Digital Initiatives Working Group at diwg@uco.edu for the permission policy on the use, reproduction or distribution of this material."],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["(Alma MMSId)9983181494502196"],"render_values":[{"text":"(Alma MMSId)9983181494502196","href":null,"code":true}]}]},"links":{"outbound_url":"https://shareok.org//handle/11244/342581","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"source_record":{"url":"https://shareok.org/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ashareok.org%3A11244%2F342581","prefix":"dim"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Creecy, James"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Haynie, Michelle L., 1975-","Porterfield, Caitlin"]},{"key":"dc:creator","label":"Author","values":["Deen, Beth A."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-05-19T14:55:14Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-05-19T14:55:14Z"]},{"key":"dc:date.issued","label":"Date","values":["2026"]},{"key":"dc:type","label":"Dc Type","values":["Academic theses"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Jackson College of Graduate Studies"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["All rights reserved by the author, who has granted UCO Chambers Library the non-exclusive right to share this material in its online repositories. Contact UCO Chambers Library's Digital Initiatives Working Group at diwg@uco.edu for the permission policy on the use, reproduction or distribution of this material."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["(Alma MMSId)9983181494502196"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://shareok.org//handle/11244/342581"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:title","label":"Title","values":["A universal method for the extraction, enrichment, and sequencing of mitochondrial genomes for the purposes of forensic wildlife investigations"]}]}],"canonical_facts":{"dc:contributor.advisor":["Creecy, James"],"dc:contributor.committeemember":["Haynie, Michelle L., 1975-","Porterfield, Caitlin"],"dc:creator":["Deen, Beth A."],"dc:date.accessioned":["2026-05-19T14:55:14Z"],"dc:date.available":["2026-05-19T14:55:14Z"],"dc:date.issued":["2026"],"dc:description.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."],"dc:identifier.other":["(Alma MMSId)9983181494502196"],"dc:identifier.uri":["https://shareok.org//handle/11244/342581"],"dc:rights":["All rights reserved by the author, who has granted UCO Chambers Library the non-exclusive right to share this material in its online repositories. Contact UCO Chambers Library's Digital Initiatives Working Group at diwg@uco.edu for the permission policy on the use, reproduction or distribution of this material."],"dc:title":["A universal method for the extraction, enrichment, and sequencing of mitochondrial genomes for the purposes of forensic wildlife investigations"],"dc:type":["Academic theses"],"thesis:institution_name":["Jackson College of Graduate Studies"]},"updated_at":"2026-08-21T16:43:16Z"}