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Microbial DNA Sequencing in Environmental Studies
KTH, School of Biotechnology (BIO). (EnvGen group)ORCID iD: 0000-0002-2025-2198
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The field of microbial ecology has just entered a new era of rapid technological development and generation of big data. The high-throughput sequencing techniques presently available provide an opportunity to extensively inventorize the blueprints of life. Now, millions of microbes of natural microbial communities can be studied simultaneously without prior cultivation. New species and new functions (genes) can be discovered just by mining sequencing data. However, there is still a tremendous number of microorganisms not yet examined, nor are the ecosystem functions these carry out. The modern genomic technologies can contribute to solve environmental problems and help us understand ecosystems, but to most efficiently do so, methods need to be continuously optimised.

 

During my Ph. D. studies, I developed a method to survey eukaryotic microbial diversity with a higher accuracy, and applied various sequencing-based approaches in an attempt to answer questions of importance in environmental research and ecology. In PAPER-I, we developed a set of 18S rRNA gene PCR primers with high taxonomic coverage, meeting the requirements of currently popular sequencing technologies and matching the richness of 18S rRNA reference sequences accumulated so far. In PAPER-II, we conducted the first sequencing-based spatial survey on the combined eukaryotic and bacterial planktonic community in the Baltic Sea to uncover the relationship of microbial diversity and environmental conditions. Here, the 18S primers designed in PAPER-I and a pair of broad-coverage 16S primers were employed to target the rRNA genes of protists and bacterioplankton for amplicon sequencing. In PAPER-III, we integrated metagenomic, metabarcoding, and metatranscriptomic data in an effort to scrutinise the protein synthesis potential (i.e., activity) of microbes in the sediment at a depth of 460 m in the Baltic Sea and, thus, disclosing microbial diversity and their possible ecological functions within such an extreme environment. Lastly, in PAPER-IV, we compared the performance of E. coli culturing, high-throughput sequencing, and portable real-time sequencing in tracking wastewater contamination in an urban stormwater system. From the aspects of cost, mobility and accuracy, we evaluated the usage of sequencing-based approaches in civil engineering, and for the first time, validated the real-time sequencing device in use within water quality monitoring.

 

In summary, these studies demonstrate how DNA sequencing of microbial communities can be applied in environmental monitoring and ecological research.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 63
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:8
Keywords [en]
DNA sequencing; Metabarcoding; Microbial ecology; Baltic Sea; Microbial community; Illumina; Oxford Nanopore; Source tracking; Stormwater
National Category
Microbiology Civil Engineering Ecology Bioinformatics and Systems Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-204897ISBN: 978-91-7729-322-4 (print)OAI: oai:DiVA.org:kth-204897DiVA, id: diva2:1086654
Public defence
2017-04-21, Air-and-Fire Lecture Hall, Tomtebodavägen 23a (Science for Life Laboratory, Stockholm), Solna, 10:00 (English)
Opponent
Supervisors
Note

Yue Hu was supported by a scholarship from the China Scholarship Council (CSC #201206950024)

Yue Hu has been publishing papers under the name "Yue O. O. Hu".

QC 20170403

Available from: 2017-04-03 Created: 2017-04-03 Last updated: 2017-04-04Bibliographically approved
List of papers
1. 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, p. 095567-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.

Keywords
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
2. Diversity of Pico- to Mesoplankton along the 2000 km Salinity Gradient of the Baltic Sea
Open this publication in new window or tab >>Diversity of Pico- to Mesoplankton along the 2000 km Salinity Gradient of the Baltic Sea
2016 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 679Article in journal (Refereed) Published
Abstract [en]

Microbial plankton form the productive base of both marine and freshwater ecosystems and are key drivers of global biogeochemical cycles of carbon and nutrients. Plankton diversity is immense with representations from all major phyla within the three domains of life. So far, plankton monitoring has mainly been based on microscopic identification, which has limited sensitivity and reproducibility, not least because of the numerical majority of plankton being unidentifiable under the light microscope. High-throughput sequencing of taxonomic marker genes offers a means to identify taxa inaccessible by traditional methods; thus, recent studies have unveiled an extensive previously unknown diversity of plankton. Here, we conducted ultra-deep Illumina sequencing (average 105 sequences/sample) of rRNA gene amplicons of surface water eukaryotic and bacterial plankton communities sampled in summer along a 2000 km transect following the salinity gradient of the Baltic Sea. Community composition was strongly correlated with salinity for both bacterial and eukaryotic plankton assemblages, highlighting the importance of salinity for structuring the biodiversity within this ecosystem. In contrast, no clear trends in alpha-diversity for bacterial or eukaryotic communities could be detected along the transect. The distribution of major planktonic taxa followed expected patterns as observed in monitoring programs, but groups novel to the Baltic Sea were also identified, such as relatives to the coccolithophore Erniliana huxleyi detected in the northern Baltic Sea. This study provides the first ultra-deep sequencing-based survey on eukaryotic and bacterial plankton biogeography in the Baltic Sea.

Place, publisher, year, edition, pages
Frontiers Media, 2016
Keywords
Baltic Sea, protists, bacterioplankton, brackish, metabarcoding, marine microbiology, microbial ecology
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-188057 (URN)10.3389/fmicb.2016.00679 (DOI)000375685400001 ()2-s2.0-84973541011 (Scopus ID)
Note

QC 20160615

Available from: 2016-06-15 Created: 2016-06-03 Last updated: 2017-11-28Bibliographically approved
3. Accumulation of DNA in an anoxic sediment – rDNA and rRNA presence of members of the microbial community from Landsort Deep, the Baltic Sea
Open this publication in new window or tab >>Accumulation of DNA in an anoxic sediment – rDNA and rRNA presence of members of the microbial community from Landsort Deep, the Baltic Sea
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Numerous investigations of bacterial communities using sequence analysis of environmental DNA have revealed extensive diversity of microbial taxa in an array of different environmental habitats. Community analysis based solely on DNA, however, does not reveal whether the detected community members are actively contributing to community functioning, or whether they are dormant or remnants of dead cells. This dilemma is of particular concern when analyzing microbial community structure of sites with a high degree of deposited matter, such as marine sediments. For example, the Baltic Sea’s deepest point, the Landsort Deep, consists of anoxic sediments with a large deposition of allochthonous organic matter from the highly stratified 460 m water column above. Our previous metagenomics results indicated the presence of potential obligately aerobic and phototrophic microorganisms in the Landsort Deep sediment. To further elucidate which taxa may contribute to ecosystem function at this site, we here present three different datasets – rDNA amplicons, rDNA reads from a shotgun metagenome and expressed rRNA from a shotgun metatranscriptome. By comparing the three datasets and the ratios between rRNA and rDNA we seek to estimate the protein synthesis potential of the community members in order to provide an indication of what taxa may have cellular activity and metabolic potential. The variation in protein synthesis potential was large, both within and between taxa, in the sediment community. Many typically anaerobic taxa, e.g. from Deltaproteobacteria and Euryarchaeota, showed a high protein synthesis potential, while typical aerobes like Flavobacteria showed a low protein synthesis potential. More surprisingly, some common Baltic Sea surface water bacteria also displayed a high protein synthesis potential, suggesting they have an active role in the anoxic sediment ecosystem at 460 m depth. Both filamentous and unicellular Cyanobacteria exhibited very high protein synthesis potential, which implies a more complex role of these bacteria in carbon cycling in the Baltic Sea than previously suggested. Moreover, Mycobacteria, that were abundant in Landsort Deep sediment metagenome compared with other marine sediment metagenomes, showed protein synthesis potentials consistent with a functional role in the sediment community. Our results provide a new window of insight into the complexities of the microbial community of Landsort Deep with implications for the understanding of other anoxic accumulation sediments.

Keywords
Sediment, Protein synthesis potential, Dormancy, Microbial activity, rDNA, rRNA, metabarcoding
National Category
Environmental Sciences Microbiology Ecology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-204662 (URN)
Note

QC 20170403

Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2017-05-03Bibliographically approved
4. Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems
Open this publication in new window or tab >>Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Urban sewer systems consist of wastewater and stormwater sewers, of which typically only

the wastewater is processed before being discharged. Occasionally, misconnections or

damages in the network occur, resulting in wastewater entering the stormwater system and

being discharged without prior processing. Cultivation of faecal indicator bacteria, such as

Escherichia coli (E. coli), is the current standard for tracing wastewater contamination. This

method is cheap but cannot be employed in the field and is characterised by its limited

specificity. Here, we compared the E. coli culturing approach with two different DNA

sequencing-based methodologies (i.e., 16S rRNA amplicon sequencing on the Illumina

MiSeq platform and shotgun metagenomic sequencing on an Oxford Nanopore MinIOn

device), analysing 73 stormwater samples collected throughout the Stockholm city areas.

High correlations were obtained between E. coli culturing counts and frequencies of human

gut microbiome sequencing reads (via amplicon sequencing), indicating that E. coli is indeed

a good indicator of faecal contamination. In contrast to E.coli culturing, amplicon sequencing

could, however, further distinguish between two different sources of contamination in an

area, where misconnections in the stormwater system were later on detected. Shotgun

metagenomic sequencing on a subset of the samples using the portable Oxford Nanopore

MinION real-time sequencing device correlated well with the amplicon sequencing data. In

summary, this study shows that DNA sequencing allows distinguishing different

contamination sources in stormwater systems and demonstrates the potential of using a

portable sequencing device in the field for tracking faecal contamination.

Keywords
stormwater, metabarcoding, oxford nanopore, illumina, microbial community, source tracking
National Category
Environmental Engineering Civil Engineering
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-204668 (URN)
Note

QC 20170403

Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2017-04-06Bibliographically approved

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