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DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies
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: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 16, 5116-5123 p.Article in journal (Refereed) Published
Abstract [en]

The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e. g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011,, and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.

Place, publisher, year, edition, pages
2014. Vol. 80, no 16, 5116-5123 p.
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-150521DOI: 10.1128/AEM.01403-14ISI: 000340038400036ScopusID: 2-s2.0-84904880610OAI: diva2:746801

QC 20140915

Available from: 2014-09-15 Created: 2014-09-05 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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