Article (Scientific journals)
Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia
Hugerth, Luisa; Muller, Emilie; Hu, Yue et al.
2014In PLoS ONE
Peer Reviewed verified by ORBi
 

Files


Full Text
Hugerth (2014)PLosONE.pdf
Publisher postprint (1.73 MB)
Download

All documents in ORBilu are protected by a user license.

Send to



Details



Abstract :
[en] High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by massively parallel sequencing technologies make the choice of sequencing region crucial for accurate phylogenetic assignments. While for 16S rDNA, relevant regions have been well described, no truly systematic design of 18S rDNA primers aimed at resolving eukaryotic diversity has yet been reported. Here we used 31,862 18S rDNA sequences to design a set of broad-taxonomic range degenerate PCR primers. We simulated the phylogenetic information that each candidate primer pair would retrieve using paired- or single-end reads of various lengths, representing different sequencing technologies. Primer pairs targeting the V4 region performed best, allowing discrimination with paired-end reads as short as 150 bp (with 75% accuracy at genus level). The conditions for PCR amplification were optimised for one of these primer pairs and this was used to amplify 18S rDNA sequences from isolates as well as from a range of environmental samples which were then Illumina sequenced and analysed, revealing good concordance between expected and observed results. In summary, the reported primer sets will allow minimally biased assessment of eukaryotic diversity in different microbial ecosystems.
Research center :
- Luxembourg Centre for Systems Biomedicine (LCSB): Eco-Systems Biology (Wilmes Group)
Disciplines :
Environmental sciences & ecology
Microbiology
Genetics & genetic processes
Biochemistry, biophysics & molecular biology
Author, co-author :
Hugerth, Luisa;  KTH Royal Institute of Technology > Science for Life Laboratory
Muller, Emilie ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
Hu, Yue;  KTH Royal Institute of Technology > Science for Life Laboratory
Lebrun, Laura ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
Roume, Hugo ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
Lundin, Daniel;  KTH Royal Institute of Technology > Science for Life Laboratory
Wilmes, Paul ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
Andersson, Anders;  KTH Royal Institute of Technology > Science for Life Laboratory
External co-authors :
yes
Language :
English
Title :
Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia
Publication date :
2014
Journal title :
PLoS ONE
ISSN :
1932-6203
Publisher :
Public Library of Science, San Franscisco, United States - California
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
The Swedish Research Councils VR [grant 2011-5689], FORMAS [grant 2009-1174] and EC BONUS project BLUEPRINT partially funded by FORMAS through grants to A.F.A, the Luxemburg National Research fund [grants ATTRACT/A09/03 and CORE/11/BM/1186762 to P.W., PHD-MARP-04 to H.R. and PRD-2011-1/SR to E.M.] and the Chinese Scholarship Council
Available on ORBilu :
since 29 May 2014

Statistics


Number of views
181 (7 by Unilu)
Number of downloads
274 (6 by Unilu)

Scopus citations®
 
174
Scopus citations®
without self-citations
164
OpenCitations
 
173
WoS citations
 
167

Bibliography


Similar publications



Contact ORBilu