Nanopore sequencing from extraction-free direct PCR of dried serum spots for portable hepatitis B virus drug-resistance typing

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dc.contributor.authorAstbury, Stuart
dc.contributor.authorNunes Soares, Marcia Maria Costa
dc.contributor.authorPeprah, Emmanuel
dc.contributor.authorKing, Barnabas
dc.contributor.authorGomes Jardim, Ana Carolina [UNESP]
dc.contributor.authorFarinha Shimizu, Jacqueline [UNESP]
dc.contributor.authorJalal, Paywast
dc.contributor.authorSaeed, Chiman H.
dc.contributor.authorSabeer, Furat T.
dc.contributor.authorIrving, William L.
dc.contributor.authorTarr, Alexander W.
dc.contributor.authorMcClure, C. Patrick
dc.contributor.institutionUniv Nottingham
dc.contributor.institutionNottingham Univ Hosp NHS Trust
dc.contributor.institutionBrazilian Minist Hlth
dc.contributor.institutionUniversidade Federal de Uberlândia (UFU)
dc.contributor.institutionUniv Sulaimani
dc.contributor.institutionHawler Med Univ
dc.contributor.institutionCent Publ Hlth Lab
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.date.accessioned2020-12-10T17:38:21Z
dc.date.available2020-12-10T17:38:21Z
dc.date.issued2020-08-01
dc.description.abstractBackground: Effective drug regimens for the treatment of hepatitis B virus (HBV) infections are essential to achieve the World Health Organisation commitment to eliminate viral hepatitis by 2030. Lamivudine (3TC) is widely used in countries with high levels of chronic HBV, however resistance has been shown to occur in up to 50 % of individuals receiving continuous monotherapy for 4 years. Telbivudine (LdT) is now more commonly used in place of lamivudine but is ineffective against 3TC-resistant HBV. Genotyping and identification of resistance-associated substitutions (RAS) is not practical in many locations. Objectives: A novel assay was designed to enable HBV genotyping and characterisation of resistance mutations directly from serum samples stored on filter paper, using Sanger and MinION sequencing. Study design: The assay was applied to a cohort of 30 samples stored on filter paper for several years with HBV viral loads ranging from 8.2 x 10(8) to 635 IU/mL. A set of 6 high-titre samples were used in a proof-of-principle study using the MinION sequencer. Results: The assay allowed determination of HBV genotype and elucidation of RAS down to 600 IU/mL using a 550bp amplicon. Sequencing of a 1.2 kb amplicon using a MinION sequencer gave results consistent with Sanger sequencing and allowed the identification of minor populations of variants. Conclusions: We present two approaches for reliable HBV sequencing and RAS identification using methods suitable for resource-limited environments. This is the first demonstration of extraction-free DNA sequencing direct from DSS using MinION and these workflows are adaptable to the investigation of other DNA viruses.en
dc.description.affiliationUniv Nottingham, Nottingham Digest Dis Ctr, Sch Med, Nottingham, England
dc.description.affiliationNottingham Univ Hosp NHS Trust, NIHR Nottingham Biomed Res Ctr, Nottingham, England
dc.description.affiliationUniv Nottingham, Nottingham, England
dc.description.affiliationUniv Nottingham, MRC EPSRC Nottingham Mol Pathol Node, Nottingham, England
dc.description.affiliationBrazilian Minist Hlth, Inst Adolfo Lutz, Sao Paulo, SP, Brazil
dc.description.affiliationUniv Nottingham, Sch Life Sci, Nottingham, England
dc.description.affiliationUniv Fed Uberlandia, Inst Biomed Sci, Uberlandia, MG, Brazil
dc.description.affiliationUniv Sulaimani, Fac Sci, Biol Dept, Sulaymaniyah, Iraq
dc.description.affiliationHawler Med Univ, Med Res Ctr, Erbil, Iraq
dc.description.affiliationCent Publ Hlth Lab, Erbil, Iraq
dc.description.affiliationSao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil
dc.description.affiliationUnespSao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil
dc.description.sponsorshipNottingham Molecular Pathology Node (United Kingdom MRC/EPSRC)
dc.description.sponsorshipNational Institute for Health Research (NIHR) Nottingham Biomedical Research Centre
dc.description.sponsorshipIdNottingham Molecular Pathology Node (United Kingdom MRC/EPSRC): MR/N005953/1
dc.format.extent7
dc.identifierhttp://dx.doi.org/10.1016/j.jcv.2020.104483
dc.identifier.citationJournal Of Clinical Virology. Amsterdam: Elsevier, v. 129, 7 p., 2020.
dc.identifier.doi10.1016/j.jcv.2020.104483
dc.identifier.issn1386-6532
dc.identifier.urihttp://hdl.handle.net/11449/195547
dc.identifier.wosWOS:000552972500012
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relation.ispartofJournal Of Clinical Virology
dc.sourceWeb of Science
dc.subjectDried blood spot
dc.subjectNanopore sequencing
dc.subjectPortable diagnostic test
dc.subjectHepatitis B virus
dc.subjectResistance-associated substitutions
dc.subjectGenotyping
dc.titleNanopore sequencing from extraction-free direct PCR of dried serum spots for portable hepatitis B virus drug-resistance typingen
dc.typeArtigo
dcterms.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dcterms.rightsHolderElsevier B.V.
unesp.author.orcid0000-0002-9844-7745[6]

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