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dc.contributor.authorKaramitros, Timokratisen
dc.contributor.authorvan Wilgenburg, Bonnieen
dc.contributor.authorWills, Marken
dc.contributor.authorKlenerman, Paulen
dc.contributor.authorMagiorkinis, Gkikasen
dc.date.accessioned2018-11-07T00:31:21Z
dc.date.available2018-11-07T00:31:21Z
dc.date.issued2018-08-02en
dc.identifier.issn1471-2164
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/284714
dc.description.abstractBackground: Human cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies. Methods: Total DNA was extracted from concentrated preparations of HCMV (strain TB40/E). The MinION libraries were constructed using the Transposase-based “Rapid Sequencing Kit”. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage, long contigs (N50= 21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50= 5,686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50= 41,056). Results: The MinION run resulted in ~47,000 reads from a single R9 flowcell and in ~100x average read depth across the virus genome. We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions. Conclusions: Third generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis.
dc.format.mediumElectronicen
dc.languageengen
dc.publisherBioMed Central
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectCell Lineen
dc.subjectHumansen
dc.subjectCytomegalovirusen
dc.subjectSequence Analysis, DNAen
dc.subjectEvolution, Molecularen
dc.subjectGenome, Viralen
dc.subjectAlgorithmsen
dc.subjectNanoporesen
dc.subjectHigh-Throughput Nucleotide Sequencingen
dc.subjectGenome Sizeen
dc.titleNanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.en
dc.typeArticle
prism.issueIdentifier1en
prism.publicationDate2018en
prism.publicationNameBMC genomicsen
prism.startingPage577
prism.volume19en
dc.identifier.doi10.17863/CAM.32086
dcterms.dateAccepted2018-07-19en
rioxxterms.versionofrecord10.1186/s12864-018-4949-6en
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2018-08-02en
dc.contributor.orcidKaramitros, Timokratis [0000-0003-0841-9159]
dc.contributor.orcidWills, Mark [0000-0001-8548-5729]
dc.identifier.eissn1471-2164
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idMRC (G0701279)
pubs.funder-project-idMRC (G9202171)
pubs.funder-project-idMRC (MR/K021087/1)


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International