{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T23:23:35Z","timestamp":1792193015560,"version":"4.4.0"},"reference-count":24,"publisher":"Microbiology Society","issue":"3","license":[{"start":{"date-parts":[[2020,3,1]],"date-time":"2020-03-01T00:00:00Z","timestamp":1583020800000},"content-version":"unspecified","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"NSW Health"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2020,3,1]]},"abstract":"\n \n \n Bordetella pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic>, the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600\u200a000 deaths are prevented annually by pertussis vaccination, but \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> infection was still responsible for over 63\u200a000 deaths globally in 2013. The re-emergence of \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic>. Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> to human DNA was determined by real-time PCR for ERV3 and IS481<\/jats:italic> (as markers of human and \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic>. However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> DNA. Sequencing read depth improved five-fold in reads to \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic>. Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of \n \n B. pertussis<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> in the era of culture-independent testing.<\/jats:p>","DOI":"10.1099\/mgen.0.000332","type":"journal-article","created":{"date-parts":[[2020,2,28]],"date-time":"2020-02-28T15:13:29Z","timestamp":1582902809000},"source":"Crossref","is-referenced-by-count":12,"title":["Optimization of sample preparation for culture-independent sequencing of Bordetella pertussis"],"prefix":"10.1099","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6483-1119","authenticated-orcid":false,"given":"Winkie","family":"Fong","sequence":"first","affiliation":[{"name":"Westmead Clinical School, The University of Sydney, Westmead, NSW, Australia"},{"name":"Centre for Infectious Diseases and Microbiology \u2013 Public Health, Westmead Hospital, Westmead, NSW, Australia"},{"name":"Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Camperdown, NSW, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4329-5198","authenticated-orcid":false,"given":"Rebecca","family":"Rockett","sequence":"additional","affiliation":[{"name":"Westmead Clinical School, The University of Sydney, Westmead, NSW, Australia"},{"name":"Centre for Infectious Diseases and Microbiology \u2013 Public Health, Westmead Hospital, Westmead, NSW, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3883-4428","authenticated-orcid":false,"given":"Verlaine","family":"Timms","sequence":"additional","affiliation":[{"name":"Centre for Infectious Diseases and Microbiology \u2013 Public Health, Westmead Hospital, Westmead, NSW, Australia"},{"name":"Westmead Clinical School, The University of Sydney, Westmead, NSW, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9261-3650","authenticated-orcid":false,"given":"Vitali","family":"Sintchenko","sequence":"additional","affiliation":[{"name":"Centre for Infectious Diseases and Microbiology \u2013 Public Health, Westmead Hospital, Westmead, NSW, Australia"},{"name":"Westmead Clinical School, The University of Sydney, Westmead, NSW, Australia"},{"name":"Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Camperdown, NSW, 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