Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.

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 ph...

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Autores principales:	Luisa W Hugerth, Emilie E L Muller, Yue O O Hu, Laura A M Lebrun, Hugo Roume, Daniel Lundin, Paul Wilmes, Anders F Andersson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c16e250c15c34385b44c4656e482720d
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spelling	oai:doaj.org-article:c16e250c15c34385b44c4656e482720d2021-11-18T08:21:54ZSystematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.1932-620310.1371/journal.pone.0095567https://doaj.org/article/c16e250c15c34385b44c4656e482720d2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24755918/?tool=EBIhttps://doaj.org/toc/1932-6203High-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.Luisa W HugerthEmilie E L MullerYue O O HuLaura A M LebrunHugo RoumeDaniel LundinPaul WilmesAnders F AnderssonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 4, p e95567 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Luisa W Hugerth Emilie E L Muller Yue O O Hu Laura A M Lebrun Hugo Roume Daniel Lundin Paul Wilmes Anders F Andersson Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.
description	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.
format	article
author	Luisa W Hugerth Emilie E L Muller Yue O O Hu Laura A M Lebrun Hugo Roume Daniel Lundin Paul Wilmes Anders F Andersson
author_facet	Luisa W Hugerth Emilie E L Muller Yue O O Hu Laura A M Lebrun Hugo Roume Daniel Lundin Paul Wilmes Anders F Andersson
author_sort	Luisa W Hugerth
title	Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.
title_short	Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.
title_full	Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.
title_fullStr	Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.
title_full_unstemmed	Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.
title_sort	systematic design of 18s rrna gene primers for determining eukaryotic diversity in microbial consortia.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/c16e250c15c34385b44c4656e482720d
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Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.

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