The Effect of Primer Choice and Short Read Sequences on the Outcome of 16S rRNA Gene Based Diversity Studies

被引:118
作者
Ghyselinck, Jonas [1 ]
Pfeiffer, Stefan [2 ]
Heylen, Kim [1 ]
Sessitsch, Angela [2 ]
De Vos, Paul [1 ]
机构
[1] Univ Ghent, Microbiol Lab, Dept Biochem & Microbiol, B-9000 Ghent, Belgium
[2] AIT Austrian Inst Technol GmbH, Dept Hlth & Environm, Bioresources Unit, Tulln, Austria
关键词
COMMUNITY STRUCTURE; SOFTWARE; TOOLS; RATES;
D O I
10.1371/journal.pone.0071360
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
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页数:14
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