Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions

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dc.contributor.authorClaesson, Marcus J.
dc.contributor.authorWang, Qiong
dc.contributor.authorO'Sullivan, Orla
dc.contributor.authorGreene-Diniz, Rachel
dc.contributor.authorCole, James R.
dc.contributor.authorRoss, R. Paul
dc.contributor.authorO'Toole, Paul W.
dc.contributor.funderDepartment of Agriculture, Food and the Marine
dc.contributor.funderU.S. Department of Agriculture
dc.contributor.funderHealth Research Board
dc.date.accessioned2017-11-14T13:24:31Z
dc.date.available2017-11-14T13:24:31Z
dc.date.issued2010
dc.description.abstractHigh-throughput molecular technologies can profile microbial communities at high resolution even in complex environments like the intestinal microbiota. Recent improvements in next-generation sequencing technologies allow for even finer resolution. We compared phylogenetic profiling of both longer (454 Titanium) sequence reads with shorter, but more numerous, paired-end reads (Illumina). For both approaches, we targeted six tandem combinations of 16S rRNA gene variable regions, in microbial DNA extracted from a human faecal sample, in order to investigate their limitations and potentials. In silico evaluations predicted that the V3/V4 and V4/V5 regions would provide the highest classification accuracies for both technologies. However, experimental sequencing of the V3/V4 region revealed significant amplification bias compared to the other regions, emphasising the necessity for experimental validation of primer pairs. The latest developments of 454 and Illumina technologies offered higher resolution compared to their previous versions, and showed relative consistency with each other. However, the majority of the Illumina reads could not be classified down to genus level due to their shorter length and higher error rates beyond 60 nt. Nonetheless, with improved quality and longer reads, the far greater coverage of Illumina promises unparalleled insights into highly diverse and complex environments such as the human gut.en
dc.description.sponsorshipUS Department of Agriculture (USDA) National Institute of Food and Agriculture (National Research Initiative (2008-35107-04542)en
dc.description.statusPeer revieweden
dc.description.versionPublished Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.articleide200
dc.identifier.citationClaesson, M. J., Wang, Q., O'Sullivan, O., Greene-Diniz, R., Cole, J. R., Ross, R. P. and O'Toole, P. W. (2010) 'Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions', Nucleic Acids Research, 38(22), e200 (13pp). doi: 10.1093/nar/gkq873en
dc.identifier.doi10.1093/nar/gkq873
dc.identifier.endpage13
dc.identifier.issn0305-1048
dc.identifier.issn1362-4962
dc.identifier.issued22
dc.identifier.journaltitleNucleic Acids Researchen
dc.identifier.startpage1
dc.identifier.urihttps://hdl.handle.net/10468/5027
dc.identifier.volume38
dc.language.isoenen
dc.publisherOxford University Pressen
dc.relation.urihttps://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkq873
dc.rights© 2010, the Authors. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.en
dc.rights.urihttp://creativecommons.org/licenses/by-nc/2.5
dc.subjectRare biosphereen
dc.subjectDiversityen
dc.subjectPrimersen
dc.titleComparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regionsen
dc.typeArticle (peer-reviewed)en
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