Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA

Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submu...

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Veröffentlicht in:Journal of microbiological methods 2015-12, Vol.119, p.239-242
Hauptverfasser: Glassing, Angela, Dowd, Scot E., Galandiuk, Susan, Davis, Brian, Jorden, Jeffrey R., Chiodini, Rodrick J.
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container_end_page 242
container_issue
container_start_page 239
container_title Journal of microbiological methods
container_volume 119
creator Glassing, Angela
Dowd, Scot E.
Galandiuk, Susan
Davis, Brian
Jorden, Jeffrey R.
Chiodini, Rodrick J.
description Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40–70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass. •Normal 16s microbiota sequencing fails to detect rare and low prevalence microbes.•LOOXSTER enrichment separates prokaryotic and eukaryotic DNA based on methylation.•On average DNA enrichment increased generated rRNA gene sequences by 3.5-fold.•DNA enrichment allowed the detection of an additional 41 bacterial genera.•New bacterial taxon accounted for as much as 25% of the total bacterial community.
doi_str_mv 10.1016/j.mimet.2015.11.001
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We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40–70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. 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subjects 16s sequencing
Bacteria - classification
Bacteria - genetics
Bacteria - isolation & purification
DNA, Bacterial - genetics
Eukaryotic human DNA
Humans
Ileum - microbiology
Metagenomics
Microbiome
Microbiota
Prokaryotic DNA
Purification
RNA, Ribosomal, 16S - genetics
Separation
title Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA
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