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High-throughput DNA Sequencingin Microbial Ecology: Methods and Applications
KTH, School of Biotechnology (BIO), Gene Technology. Science for Life Laboratory. (Environmental genomics)ORCID iD: 0000-0001-5432-1764
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.
Series
TRITA-BIO-Report, ISSN 1654-2312
Keyword [en]
Microbial ecology; Baltic Sea; Next-generation sequencing; Amplicon sequencing; Metagenomics
National Category
Microbiology Ecology Bioinformatics and Systems Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-186162ISBN: 978-91-7595-967-2 (print)OAI: oai:DiVA.org:kth-186162DiVA: diva2:925868
Public defence
2016-05-27, Farmakologi salen, Karolinska instituet, Nobelsväg 2, Solna, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 2011-5689
Note

QC 20150505

Available from: 2016-05-04 Created: 2016-05-03 Last updated: 2016-05-04Bibliographically approved
List of papers
1. DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies
Open this publication in new window or tab >>DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies
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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, http://dx.doi.org/10.1038/ismej.2011.41), 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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-150521 (URN)10.1128/AEM.01403-14 (DOI)000340038400036 ()2-s2.0-84904880610 (Scopus ID)
Note

QC 20140915

Available from: 2014-09-15 Created: 2014-09-05 Last updated: 2017-12-05Bibliographically approved
2. Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia
Open this publication in new window or tab >>Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

Keyword
Human Gut Microbiota, Bacterial Communities, Species Richness, Water, Dynamics, Rdna, Identification, Generation, Magnitude, Fragments
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-145827 (URN)10.1371/journal.pone.0095567 (DOI)000335240300054 ()2-s2.0-84899679811 (Scopus ID)
Funder
Swedish Research Council, 2011-5689Formas, 2009-1174Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140604

Available from: 2014-06-04 Created: 2014-06-02 Last updated: 2017-12-05Bibliographically approved
3. Seasonal dynamics and interactions among Baltic Sea prokaryoticand eukaryotic plankton assemblages
Open this publication in new window or tab >>Seasonal dynamics and interactions among Baltic Sea prokaryoticand eukaryotic plankton assemblages
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

One of the main goals of microbial ecology is to identify the mechanismsthat regulate patterns in community structure at temporal scalescompatible with populations’ turnover times across complete seasonalcycles. Here, we examined high-frequency temporal dynamics of marineplankton from a sampling effort covering 2011-2013, roughly twice weekly,comprising 144 samples. Bacterial and eukaryotic communities wereprofiled by 16S and 18S high-throughput sequencing, respectively.Interestingly, we found that no combination of the measured environmentalparameters could predict a significant proportion of the variation inpopulation dynamics of bacterioplankton, and even less so for eukaryoticplankton. Large differences in physicochemical conditions and communitycomposition typical of temperate climates mean that different regimes canquickly succeed each other over the year, with the relative importance ofdifferent drivers changing equally rapidly. Nevertheless, our approachrevealed interesting recurrent co-occurrence patterns across distinctenvironmental changes. Hence, we could make abundance predictions formore than half of the most frequent OTUs based on interactions with otherOTUs. These results suggests that a complex set of biotic interactions arecontributing to temporal patterns among planktonic assemblages despiterapid changes in environmental conditions.

Keyword
Microbiology; Plankton; Baltic Sea; Environmental modelling; Food web; Network
National Category
Microbiology Ecology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-186160 (URN)
Funder
Swedish Research Council, 2011-5689Swedish Research Council Formas, ECOCHANGE
Note

QC 20160504

Available from: 2016-05-03 Created: 2016-05-03 Last updated: 2016-05-04Bibliographically approved
4. Metagenome-assembled genomes uncover a global brackish microbiome
Open this publication in new window or tab >>Metagenome-assembled genomes uncover a global brackish microbiome
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2015 (English)In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 16, 279Article in journal (Refereed) Published
Abstract [en]

Background: Microbes are main drivers of biogeochemical cycles in oceans and lakes. Although the genome is a foundation for understanding the metabolism, ecology and evolution of an organism, few bacterioplankton genomes have been sequenced, partly due to difficulties in cultivating them. Results: We use automatic binning to reconstruct a large number of bacterioplankton genomes from a metagenomic time-series from the Baltic Sea, one of world's largest brackish water bodies. These genomes represent novel species within typical freshwater and marine clades, including clades not previously sequenced. The genomes' seasonal dynamics follow phylogenetic patterns, but with fine-grained lineage-specific variations, reflected in gene-content. Signs of streamlining are evident in most genomes, and estimated genome sizes correlate with abundance variation across filter size fractions. Comparing the genomes with globally distributed metagenomes reveals significant fragment recruitment at high sequence identity from brackish waters in North America, but little from lakes or oceans. This suggests the existence of a global brackish metacommunity whose populations diverged from freshwater and marine relatives over 100,000 years ago, long before the Baltic Sea was formed (8000 years ago). This markedly contrasts to most Baltic Sea multicellular organisms, which are locally adapted populations of freshwater or marine counterparts. Conclusions: We describe the gene content, temporal dynamics and biogeography of a large set of new bacterioplankton genomes assembled from metagenomes. We propose that brackish environments exert such strong selection that lineages adapted to them flourish globally with limited influence from surrounding aquatic communities.

Place, publisher, year, edition, pages
BioMed Central, 2015
Keyword
Metagenome, Bacterioplankton, Ecology, Evolution, Marine, Brackish, Baltic Sea
National Category
Genetics Microbiology
Identifiers
urn:nbn:se:kth:diva-180496 (URN)10.1186/s13059-015-0834-7 (DOI)000366898100001 ()2-s2.0-84949761326 (Scopus ID)
Note

QC 20160118

Available from: 2016-01-18 Created: 2016-01-14 Last updated: 2017-11-30Bibliographically approved

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