Primer and platform effects on 16S rRNA tag sequencing

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DOIResolve DOI: http://doi.org/10.3389/fmicb.2015.00771
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TypeArticle
Journal titleFrontiers in Microbiology
ISSN1664-302X
Volume6
IssueAUG
Article number771
Subject16S rRNA gene sequencing; microbial population and community ecology; high throughput sequencing; microbial diversity; community assembly; amplification; sequencing error
AbstractSequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6-V8, and V7-V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. Beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.
Publication date
LanguageEnglish
AffiliationEnergy, Mining and Environment; National Research Council Canada
Peer reviewedYes
NPARC number21276978
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Record identifier31d6d642-e2e7-481d-b46d-855cb2ebf458
Record created2015-11-10
Record modified2016-05-09
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