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dc.contributor.authorMcGovern, Emily
dc.contributor.authorBlackshields, Gordon
dc.contributor.authorMcCabe, Matthew
dc.contributor.authorWaters, Sinead M.
dc.date.accessioned2019-07-16T16:12:59Z
dc.date.available2019-07-16T16:12:59Z
dc.date.issued2018-06-25
dc.identifier.citationMcGovern E, Waters SM, Blackshields G and McCabe MS (2018) Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations. Front. Microbiol. 9:1365. doi: 10.3389/fmicb.2018.01365en_US
dc.identifier.urihttp://hdl.handle.net/11019/1673
dc.descriptionpeer-revieweden_US
dc.description.abstractThe rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICSTM). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3–V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman’s correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol.en_US
dc.description.sponsorshipThe study was funded by a FACCE-JPI “Rumen Stability” grant (2014). EM was funded by a Walsh Fellowship award (Ref. No. 2014231).
dc.language.isoenen_US
dc.publisherFrontiersen_US
dc.relation.ispartofseriesFrontiers in Microbiology;
dc.rightsAttribution-NonCommercial-ShareAlike 3.0 United States*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/3.0/us/*
dc.subjectmock communityen_US
dc.subjectrumenen_US
dc.subjectampliconen_US
dc.subjectPCRen_US
dc.subjectmicrobiotaen_US
dc.subjectphylogenetic analysisen_US
dc.titleEvaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populationsen_US
dc.typeArticleen_US
dc.identifier.doihttps://dx.doi.org/10.3389/fmicb.2018.01365
dc.contributor.sponsorFACCE-JPI granten_US
dc.contributor.sponsorWalsh Fellowship awarden_US
dc.contributor.sponsorGrantNumber2014231en_US
refterms.dateFOA2019-07-16T16:12:59Z


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