{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T15:41:00Z","timestamp":1792510860435,"version":"4.5.0"},"reference-count":52,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"France 2030","award":["ANR-22-PAMR-0009"],"award-info":[{"award-number":["ANR-22-PAMR-0009"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,10,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Clostridioides difficile<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Clostridium perfringens<\/jats:italic>\n                      are Gram-positive, spore-forming anaerobic pathogens affecting humans and animals, for which genomic data have been mainly generated using short-read or hybrid sequencing approaches. In this study, we evaluated three commercial non-bead-beating DNA extraction kits designed for high-molecular-weight DNA recovery for Oxford Nanopore long-read whole-genome sequencing of two\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      and two\n                      <jats:italic toggle=\"yes\">C. perfringens<\/jats:italic>\n                      strains, including one reference strain and one clinical or environmental isolate per species. Based on sequencing performance and kit ease of use, one kit was selected for additional sequencing of plasmid-carrying strains of both species. All three kits allowed correct identification of sequence types, toxin-encoding genes, and antimicrobial resistance determinants, confirming their suitability for clinical and epidemiological applications. However, the BT MasterPure Kit provided the highest DNA concentrations, longest fragment sizes, and superior read lengths and N50 values, particularly for\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      , achieving &gt;100\u00d7 coverage and enabling reliable circularization of chromosomes and plasmids, including a\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      metronidazole resistance plasmid and\n                      <jats:italic toggle=\"yes\">C. perfringens<\/jats:italic>\n                      plasmids carrying toxin and antibiotic resistance genes. The other kits produced slightly lower DNA yields, resulting in shorter reads and reduced genome coverage for\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      , highlighting the challenge of extracting high-quality DNA from Gram-positive, spore-forming bacteria. Overall, this study provides practical guidance for selecting DNA extraction protocols optimized for Oxford Nanopore sequencing of\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">C. perfringens<\/jats:italic>\n                      , supporting high-quality genome assemblies and plasmid characterization and facilitating the routine genomic surveillance of clinically relevant spore-forming pathogens.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      High-quality genomic data are essential for accurate characterization of\n                      <jats:italic toggle=\"yes\">Clostridioides difficile<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Clostridium perfringens<\/jats:italic>\n                      , two clinically and epidemiologically important Gram-positive, spore-forming pathogens. However, long-read sequencing performance can be strongly influenced by the choice of DNA extraction method, particularly for organisms with robust cell walls, where commonly used methods can lead to fragmented DNA. In this work, DNA of four strains was extracted using three commercial high-molecular-weight DNA extraction kits and sequenced using Oxford Nanopore Technologies. The best-performing kit was also evaluated using three additional strains known to harbor plasmids in order to assess its plasmid recovery efficiency. The results demonstrated successful plasmid recovery, circularization, and characterization. DNA extraction protocols optimized for Oxford Nanopore sequencing enable the rapid and cost-effective characterization of\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">C. perfringens<\/jats:italic>\n                      for genomic surveillance or outbreak investigations.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02407-26","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T13:01:31Z","timestamp":1789131691000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Comparative evaluation of three high-molecular-weight DNA extraction kits for Oxford Nanopore sequencing of\n                    <i>Clostridioides difficile<\/i>\n                    and\n                    <i>Clostridium perfringens<\/i>"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-2128-9126","authenticated-orcid":true,"given":"Alexandre","family":"Mrozinski","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00g700j37","id-type":"ROR","asserted-by":"publisher"}],"name":"Agro\u00e9cologie Laboratory, INRAE, Universit\u00e9 Bourgogne Europe","place":["Dijon, France"]},{"name":"ANSES, Laboratoire de Ploufragan-Plouzan\u00e9-Niort, Unit\u00e9 HQPAP, BP53","place":["Ploufragan, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7310-9977","authenticated-orcid":false,"given":"Daniel","family":"Martak","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00g700j37","id-type":"ROR","asserted-by":"publisher"}],"name":"Agro\u00e9cologie Laboratory, INRAE, Universit\u00e9 Bourgogne Europe","place":["Dijon, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6424-5176","authenticated-orcid":false,"given":"Guillaume","family":"Reboul","sequence":"additional","affiliation":[{"name":"ANSES, Laboratoire de Ploufragan-Plouzan\u00e9-Niort, Unit\u00e9 HQPAP, BP53","place":["Ploufragan, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2677-9521","authenticated-orcid":false,"given":"Muriel","family":"Ehmig","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Clostridioides difficile Saint-Antoine Hospital, AP-HP","place":["Paris, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9670-3274","authenticated-orcid":false,"given":"Laure","family":"Diancourt","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0495fxg12","id-type":"ROR","asserted-by":"publisher"}],"name":"National Reference Laboratory for Anaerobic Bacteria and Botulism, Institut Pasteur","place":["Paris, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4165-0670","authenticated-orcid":false,"given":"Fr\u00e9d\u00e9ric","family":"Barbut","sequence":"additional","affiliation":[{"name":"National Reference Laboratory for Clostridioides difficile Saint-Antoine Hospital, AP-HP","place":["Paris, France"]},{"id":[{"id":"https:\/\/ror.org\/05f82e368","id-type":"ROR","asserted-by":"publisher"}],"name":"INSERM 1139, FPRM, Universit\u00e9 Paris Cit\u00e9","place":["Paris, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1970-3801","authenticated-orcid":false,"given":"Caroline","family":"Le Mar\u00e9chal","sequence":"additional","affiliation":[{"name":"ANSES, Laboratoire de Ploufragan-Plouzan\u00e9-Niort, Unit\u00e9 HQPAP, BP53","place":["Ploufragan, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8273-9372","authenticated-orcid":true,"given":"Edward","family":"Topp","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00g700j37","id-type":"ROR","asserted-by":"publisher"}],"name":"Agro\u00e9cologie Laboratory, INRAE, Universit\u00e9 Bourgogne Europe","place":["Dijon, France"]},{"id":[{"id":"https:\/\/ror.org\/02grkyz14","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, University of Western Ontario","place":["London, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2164"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.disamonth.2015.01.006"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2018.01.011"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-micro-090816-093458"},{"key":"e_1_3_4_6_2","unstructured":"CDC. 2024. 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