Comparing apples and oranges?: Next generation sequencing and its impact on microbiome analysis

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dc.contributor.author Clooney, Adam G.
dc.contributor.author Fouhy, Fiona
dc.contributor.author Sleator, Roy D.
dc.contributor.author O'Driscoll, Aisling
dc.contributor.author Stanton, Catherine
dc.contributor.author Cotter, Paul D.
dc.contributor.author Claesson, Marcus J.
dc.date.accessioned 2017-02-20T15:56:32Z
dc.date.available 2017-02-20T15:56:32Z
dc.date.issued 2016-02-05
dc.identifier.citation Clooney, A. G., Fouhy, F., Sleator, R. D., O’ Driscoll, A., Stanton, C., Cotter, P. D. and Claesson, M. J. (2016) 'Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis', PLOS ONE, 11(2), pp. e0148028. doi:10.1371/journal.pone.0148028 en
dc.identifier.volume 11 en
dc.identifier.issued 2 en
dc.identifier.startpage e0148028 en
dc.identifier.issn 1932-6203
dc.identifier.uri http://hdl.handle.net/10468/3654
dc.identifier.doi 10.1371/journal.pone.0148028
dc.description.abstract Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance in microbiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced with MiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10 million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques. en
dc.description.sponsorship Science Foundation Ireland (SFI under Grant Number SFI/12/RC/2273 and 11/PI/1137) en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher Public Library of Science en
dc.rights © 2016 The Authors. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. en
dc.rights.uri https://creativecommons.org/publicdomain/zero/1.0/ en
dc.subject Shotgun sequencing en
dc.subject Microbiome en
dc.subject Ribosomal RNA en
dc.subject Sequence assembly tools en
dc.subject Gene sequencing en
dc.subject Polymerase chain reaction en
dc.subject Taxonomy en
dc.subject Microbial taxonomy en
dc.title Comparing apples and oranges?: Next generation sequencing and its impact on microbiome analysis en
dc.type Article (peer-reviewed) en
dc.internal.authorcontactother Marcus Claesson, Microbiology, University College Cork, Cork, Ireland. +353-21-490-3000 Email: m.claesson@ucc.ie en
dc.internal.availability Full text available en
dc.date.updated 2017-02-20T15:49:17Z
dc.description.version Published Version en
dc.internal.rssid 384309046
dc.internal.rssid 384309046
dc.contributor.funder Science Foundation Ireland en
dc.contributor.funder European Commission en
dc.contributor.funder Seventh Framework Programme en
dc.description.status Peer reviewed en
dc.identifier.journaltitle Plos One en
dc.internal.copyrightchecked No !!CORA!! en
dc.internal.licenseacceptance Yes en
dc.internal.IRISemailaddress m.claesson@ucc.ie en
dc.internal.IRISemailaddress aisling.odriscoll@ucc.ie en
dc.relation.project info:eu-repo/grantAgreement/EC/FP7::SP1::HEALTH/603038/EU/Cystic Fibrosis Microbiome-determined Antibiotic Therapy Trial in Exacerbations: Results Stratified./CFMATTERS en


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© 2016 The Authors. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Except where otherwise noted, this item's license is described as © 2016 The Authors. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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