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Bioinformatic Methods in Metagenomics
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. (Environmental Genomics)
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microbial organisms are a vital part of our global ecosystem. Yet, our knowledge of them is still lacking. Direct sequencing of microbial communities, i.e. metagenomics, have enabled detailed studies of these microscopic organisms by inspection of their DNA sequences without the need to culture them. Furthermore, the development of modern high- throughput sequencing technologies have made this approach more powerful and cost-effective. Taken together, this has shifted the field of microbiology from previously being centered around microscopy and culturing studies, to largely consist of computational analyses of DNA sequences. One such computational analysis which is the main focus of this thesis, aims at reconstruction of the complete DNA sequence of an organism, i.e. its genome, directly from short metagenomic sequences.

This thesis consists of an introduction to the subject followed by five papers. Paper I describes a large metagenomic data resource spanning the Baltic Sea microbial communities. This dataset is complemented with a web-interface allowing researchers to easily extract and visualize detailed information. Paper II introduces a bioinformatic method which is able to reconstruct genomes from metagenomic data. This method, which is termed CONCOCT, is applied on Baltic Sea metagenomics data in Paper III and Paper V. This enabled the reconstruction of a large number of genomes. Analysis of these genomes in Paper III led to the proposal of, and evidence for, a global brackish microbiome. Paper IV presents a comparison between genomes reconstructed from metagenomes with single-cell sequenced genomes. This further validated the technique presented in Paper II as it was found to produce larger and more complete genomes than single-cell sequencing.

Abstract [sv]

Mikrobiella organismer är en vital del av vårt globala ekosystem. Trots detta är vår kunskap om dessa fortfarande begränsad. Sekvensering direkt applicerad på mikrobiella samhällen, så kallad metagenomik, har möjliggjort detaljerade studier av dessa mikroskopiska organismer genom deras DNA-sekvenser. Utvecklingen av modern sekvenseringsteknik har vidare gjort denna strategi både mer kraftfull och mer kostnadseffektiv. Sammantaget har detta förändrat mikrobiologi-fältet, från att ha varit centrerat kring mikroskopi, till att till stor del bero på dataintensiva analyser av DNA-sekvenser. En sådan analys, som är det huvudsakliga fokuset för den här avhandlingen, syftar till att återskapa den kompletta DNA-sekvensen för en organism, dvs. dess genom, direkt från korta metagenom-sekvenser.

Den här avhandlingen består av en introduktion till ämnet, följt av fem artiklar. Artikel I beskriver en omfattande databas för metagenomik över Östersjöns mikrobiella samhällen. Till denna databas hör också en webbsida som ger forskare möjlighet att lätt extrahera och visualisera detaljerad information. Artikel II introducerar en bioinformatisk metod som kan återskapa genom från metagenom. Denna metod, som kallas CONCOCT, används för data från Östersjön i artikel III och Artikel V. Detta möjliggjorde återskapandet av ett stort antal genom. Analys av dessa genom presenterad i Artikel III ledde till hypotesen om, och belägg för, ett globalt brackvattenmikrobiom. Artikel IV innehåller en jämförelse mellan genom återskapade från metagenom och individuellt sekvenserade genom. Detta validerade metoden som presenterades i Artikel II ytterligare då denna metod visade sig producera större och mer kompletta genom än sekvensering av individuella celler.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2018. , p. 51
Series
TRITA-CBH-FOU ; 2018:25
Keywords [en]
Bioinformatics, Metagenomics, Microbiome, Binning, Baltic Sea
Keywords [sv]
Bioinformatik, Metagenomik, Mikrobiom, Binning, Östersjön
National Category
Bioinformatics and Systems Biology Bioinformatics (Computational Biology) Microbiology
Identifiers
URN: urn:nbn:se:kth:diva-227965ISBN: 978-91-7729-799-4 (print)OAI: oai:DiVA.org:kth-227965DiVA, id: diva2:1206022
Public defence
2018-06-08, Air and Fire, Science for Life Laboratory, Tomtebodavägen 23, Solna, 10:00 (English)
Opponent
Supervisors
Funder
BONUS - Science for a better future of the Baltic Sea region, Art 185
Note

QC 20180516

Available from: 2018-05-16 Created: 2018-05-15 Last updated: 2018-05-16Bibliographically approved
List of papers
1. BARM and BalticMicrobeDB, a reference metagenome and interface to meta-omic data for the Baltic Sea
Open this publication in new window or tab >>BARM and BalticMicrobeDB, a reference metagenome and interface to meta-omic data for the Baltic Sea
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The Baltic Sea is one of the world’s largest brackish water bodies and is characterised by pronounced physicochemical gradients where microbes are the main biogeochemical catalysts. Meta-omic methods provide rich information on the composition of, and activities within microbial ecosystems, but are computationally heavy to perform. We here present the BAltic Sea Reference Metagenome (BARM), complete with annotated genes to facilitate further studies with much less computational effort. The assembly is constructed using 2.6 billion metagenomic reads from 81 water samples, spanning both spatial and temporal dimensions, and contains 6.8 million genes that have been annotated for function and taxonomy. The assembly is useful as a reference, facilitating taxonomic and functional annotation of additional samples by simply mapping their reads against the assembly. This capability is demonstrated by the successful mapping and annotation of 24 external samples. In addition, we present a public web interface, BalticMicrobeDB, for interactive exploratory analysis of the dataset.

National Category
Bioinformatics and Systems Biology Bioinformatics (Computational Biology) Microbiology Ecology
Identifiers
urn:nbn:se:kth:diva-227959 (URN)
Funder
BONUS - Science for a better future of the Baltic Sea region, Art 185Swedish Research Council, 2011-5689
Note

QC 20180516

Available from: 2018-05-15 Created: 2018-05-15 Last updated: 2019-06-12Bibliographically approved
2. Binning metagenomic contigs by coverage and composition
Open this publication in new window or tab >>Binning metagenomic contigs by coverage and composition
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2014 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 11, no 11, p. 1144-1146Article in journal (Refereed) Published
Abstract [en]

Shotgun sequencing enables the reconstruction of genomes from complex microbial communities, but because assembly does not reconstruct entire genomes, it is necessary to bin genome fragments. Here we present CONCOCT, a new algorithm that combines sequence composition and coverage across multiple samples, to automatically cluster contigs into genomes. We demonstrate high recall and precision on artificial as well as real human gut metagenome data sets.

Keywords
Sequences, Genomes
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-157613 (URN)10.1038/NMETH.3103 (DOI)000344580600021 ()25218180 (PubMedID)2-s2.0-84908597294 (Scopus ID)
Funder
Swedish Research Council, 2011-5689Formas, 2009-1174Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20141212

Available from: 2014-12-12 Created: 2014-12-11 Last updated: 2018-05-15Bibliographically approved
3. 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, article id 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
Keywords
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: 2018-05-15Bibliographically approved
4. Genomes from uncultivated prokaryotes: a comparison of metagenome-assembled and single-amplified genomes
Open this publication in new window or tab >>Genomes from uncultivated prokaryotes: a comparison of metagenome-assembled and single-amplified genomes
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Prokaryotes dominate the biosphere and regulate biogeochemical processes essential to all life. Yet, our knowledge about their biology is for the most part limited to the minority that has been successfully cultured. Molecular techniques now allow for obtaining genome sequences of uncultivated prokaryotic taxa, facilitating in-depth analyses that may ultimately improve our understanding of these key organisms.

Results: We compared results from two culture-independent strategies for recovering bacterial genomes: single-amplified genomes and metagenome-assembled genomes. Single-amplified genomes were obtained from samples collected at an offshore station in the Baltic Sea Proper and compared to previously obtained metagenome-assembled genomes from a time series at the same station. Among 16 single-amplified genomes analyzed, seven were found to match metagenome-assembled genomes, affiliated with a diverse set of taxa. Notably, genome pairs between the two approaches were nearly identical (>98.7% identity) across overlapping regions (30-80% of each genome). Within matching pairs, the single-amplified genomes were consistently smaller and less complete, whereas the genetic functional profiles were maintained. For the metagenome-assembled genomes, only on average 3.6% of the bases were estimated to be missing from the genomes due to wrongly binned contigs; the metagenome assembly was found to cause incompleteness to a higher degree than the binning procedure.

Conclusions: The strong agreement between the single-amplified and metagenome-assembled genomes emphasizes that both methods generate accurate genome information from uncultivated bacteria. Importantly, this implies that the research questions and the available resources are allowed to determine the selection of genomics approach for microbiome studies.

Keywords
Single-amplified genomes, Metagenome-assembled genomes, Metagenomics, Binning, Single-cell genomics
National Category
Bioinformatics and Systems Biology Microbiology
Identifiers
urn:nbn:se:kth:diva-227946 (URN)
Funder
Swedish Research Council, 2011-4369Swedish Research Council, 2011-5689BONUS - Science for a better future of the Baltic Sea region, 185Swedish Research Council FormasEU, European Research Council, 310039-PUZZLE_CELLSwedish Foundation for Strategic Research , SSF-FFL5Swedish Research Council, 2015-04959
Note

QC 20180516

Available from: 2018-05-15 Created: 2018-05-15 Last updated: 2018-05-16Bibliographically approved
5. Recovering 2,032 Baltic Sea microbial genomes by optimized metagenomic binning
Open this publication in new window or tab >>Recovering 2,032 Baltic Sea microbial genomes by optimized metagenomic binning
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Aquatic microorganism are key drivers of global biogeochemical cycles and form the basis of aquatic food webs. However, there is still much left to be learned about these organisms and their interaction within specific environments, such as the Baltic Sea. Crucial information for such an understanding can be found within the genome sequences of organisms within the microbial community.

In this study, the previous set of Baltic Sea clusters, constructed by Hugert et al., is greatly expanded using a large set of metagenomic samples, spanning the environmental gradients of the Baltic Sea. In total, 124 samples were individually assembled and binned to obtain 2,032 Metagenome Assembled Genomes (MAGs), clustered into 353 prokaryotic and 14 eukaryotic species- level clusters. The prokaryotic genomes were widely distributed over the prokaryotic tree of life, representing 20 different phyla, while the eukaryotic genomes were mostly limited to the division of Chlorophyta. The large number of reconstructed genomes allowed us to identify key factors determining the quality of the genome reconstructions.

The Baltic Sea is heavily influenced of human activities of which we might not see the full implications. The genomes reported within this study will greatly aid further studies in our strive for an understanding of the Baltic Sea microbial ecosystem.

National Category
Bioinformatics and Systems Biology Microbiology
Identifiers
urn:nbn:se:kth:diva-227963 (URN)
Funder
BONUS - Science for a better future of the Baltic Sea region, Art 185Swedish Research Council, 2011-5689
Note

QC 20180516

Available from: 2018-05-15 Created: 2018-05-15 Last updated: 2018-05-16Bibliographically approved

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Alneberg, Johannes

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