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The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies

(2013) PLOS ONE. 8(8).
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Abstract
Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment.
Keywords
MAXIMUM-LIKELIHOOD, SPECIES LIVING TREE, ENVIRONMENTAL SURVEYS, PHYLOGENETIC ANALYSES, COMMUNITY STRUCTURE, SOFTWARE, BACTERIA, TOOLS, RATES, ARB

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Citation

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Chicago
Ghyselinck, Jonas, Stefan Pfeiffer, Kim Heylen, Angela Sessitsch, and Paul De Vos. 2013. “The Effect of Primer Choice and Short Read Sequences on the Outcome of 16S rRNA Gene Based Diversity Studies.” Plos One 8 (8).
APA
Ghyselinck, J., Pfeiffer, S., Heylen, K., Sessitsch, A., & De Vos, P. (2013). The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies. PLOS ONE, 8(8).
Vancouver
1.
Ghyselinck J, Pfeiffer S, Heylen K, Sessitsch A, De Vos P. The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies. PLOS ONE. 2013;8(8).
MLA
Ghyselinck, Jonas, Stefan Pfeiffer, Kim Heylen, et al. “The Effect of Primer Choice and Short Read Sequences on the Outcome of 16S rRNA Gene Based Diversity Studies.” PLOS ONE 8.8 (2013): n. pag. Print.
@article{4135072,
  abstract     = {Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment.},
  articleno    = {e71360},
  author       = {Ghyselinck, Jonas and Pfeiffer, Stefan and Heylen, Kim and Sessitsch, Angela and De Vos, Paul},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  keyword      = {MAXIMUM-LIKELIHOOD,SPECIES LIVING TREE,ENVIRONMENTAL SURVEYS,PHYLOGENETIC ANALYSES,COMMUNITY STRUCTURE,SOFTWARE,BACTERIA,TOOLS,RATES,ARB},
  language     = {eng},
  number       = {8},
  pages        = {14},
  title        = {The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies},
  url          = {http://dx.doi.org/10.1371/journal.pone.0071360},
  volume       = {8},
  year         = {2013},
}

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