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Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 4, 095567- p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 4, 095567- p.
Keyword [en]
Human Gut Microbiota, Bacterial Communities, Species Richness, Water, Dynamics, Rdna, Identification, Generation, Magnitude, Fragments
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-145827DOI: 10.1371/journal.pone.0095567ISI: 000335240300054ScopusID: 2-s2.0-84899679811OAI: diva2:721370
Swedish Research Council, 2011-5689Formas, 2009-1174Science for Life Laboratory - a national resource center for high-throughput molecular bioscience

QC 20140604

Available from: 2014-06-04 Created: 2014-06-02 Last updated: 2016-05-04Bibliographically approved
In thesis
1. High-throughput DNA Sequencingin Microbial Ecology: Methods and Applications
Open this publication in new window or tab >>High-throughput DNA Sequencingin Microbial Ecology: Methods and Applications
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microorganisms play central roles in planet Earth’s geochemical cycles, in food production, and in health and disease of humans and livestock. In spite of this, most microbial life forms remain unknown and unnamed, their ecological importance and potential technological applications beyond the realm of speculation. This is due both to the magnitude of microbial diversity and to technological limitations. Of the many advances that have enabled microbiology to reach new depth and breadth in the past decade, one of the most important is affordable high-throughput DNA sequencing. This technology plays a central role in each paper in this thesis.

Papers I and II are focused on developing methods to survey microbial diversity based on marker gene amplification and sequencing. In Paper I we proposed a computational strategy to design primers with the highest coverage among a given set of sequences and applied it to drastically improve one of the most commonly used primer pairs for ecological surveys of prokaryotes. In Paper II this strategy was applied to an eukaryotic marker gene. Despite their importance in the food chain, eukaryotic microbes are much more seldom surveyed than bacteria. Paper II aimed at making this domain of life more amenable to high-throughput surveys.

In Paper III, the primers designed in papers I and II were applied to water samples collected up to twice weekly from 2011 to 2013 at an offshore station in the Baltic proper, the Linnaeus Microbial Observatory. In addition to tracking microbial communities over these three years, we created predictive models for hundreds of microbial populations, based on their co-occurrence with other populations and environmental factors.

In paper IV we explored the entire metagenomic diversity in the Linnaeus Microbial Observatory. We used computational tools developed in our group to construct draft genomes of abundant bacteria and archaea and described their phylogeny, seasonal dynamics and potential physiology. We were also able to establish that, rather than being a mixture of genomes from fresh and saline water, the Baltic Sea plankton community is composed of brackish specialists which diverged from other aquatic microorganisms thousands of years before the formation of the Baltic itself.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. x, 46 p.
TRITA-BIO-Report, ISSN 1654-2312
Microbial ecology; Baltic Sea; Next-generation sequencing; Amplicon sequencing; Metagenomics
National Category
Microbiology Ecology Bioinformatics and Systems Biology
Research subject
urn:nbn:se:kth:diva-186162 (URN)978-91-7595-967-2 (ISBN)
Public defence
2016-05-27, Farmakologi salen, Karolinska instituet, Nobelsväg 2, Solna, 09:15 (English)
Swedish Research Council, 2011-5689

QC 20150505

Available from: 2016-05-04 Created: 2016-05-03 Last updated: 2016-05-04Bibliographically approved

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