Identifying prokaryotic consortia that live in close interaction with algae

136 Pages·2015·3.22 MB·English

Identifying prokaryotic consortia that live in close
interaction with algae

Assia SALTYKOVA

Master’s dissertation submitted to obtain the degree of
Master of Science in Biochemistry and Biotechnology
Major Bioinformatics and Systems Biology
Academic year 2014-2015

Promoter: Prof. Dr. Kathleen Marchal
Scientific supervisors: Stephane Rombauts and Sergio Pulido Tamayo
UGent - Department Information Technology
UGent - Department Plant Biotechnology and Bioinformatics
VIB - Department Plant Systems Biology
Research Group Data Integration and Biological Networks

Acknowledgements
Acknowledgements
It is a great pleasure to thank those who made this work possible. I am grateful to Prof. Dr.
Kathleen Marshal for giving me the opportunity to make this thesis and to Sergio Pulido for
valuable advices and for revising the manuscript. Special thanks go to Stephane Rombauts who
has provided the data and who with endless patience has guided me through the practical part
of this work. It was also an adorable experience to join the Biocomp group with its absolutely
unique atmosphere and people.

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Acknowledgements

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Table of contents
Table of contents
Acknowledgements .............................................................................................................. i
Table of contents ................................................................................................................ iii
List of abbreviations .......................................................................................................... vii
Samenvatting ..................................................................................................................... ix
Abstract ............................................................................................................................. xi
1. Introduction .................................................................................................................... 1
1.1 Algae and the associated bacteria. ............................................................................. 3
1.1.1 Beneficial interactions between algae and bacteria. .................................................. 4
1.1.2 Detrimental interactions and defense. ........................................................................ 6
1.1.3 Structure of algal-associated bacterial communities. ................................................. 7
1.1.4 Future perspectives. ..................................................................................................... 9
1.2 Studying algal-bacterial interactions using whole-genome sequencing data. ............. 10
1.2.1 Illumina sequencing and NGS data assembly. ........................................................... 11
1.2.2 Binning of the data. ................................................................................................... 13
1.2.3 Using CONCOCT for binning of algal-bacterial assemblies. ...................................... 17
1.2.4 Estimating cluster quality. ......................................................................................... 18
2. Aim ............................................................................................................................... 21
3. Results .......................................................................................................................... 23
3.1 Assembly of non-algal reads within O. tauri sequencing data and CONCOCT-assisted
binning. ......................................................................................................................... 24
3.2 Assessing the possibility to better delineate the eukaryote target genome from
contaminants. ............................................................................................................... 28
3.3 Binning of O. mediterraneus data. ............................................................................ 29
3.4 Binning of filtered P. crispa assembly. ...................................................................... 31
3.5 Binning of C. braunii data ......................................................................................... 35
3.4.1 Binning of German C. braunii assembly .................................................................... 36
3.4.2 Binning of Japanese C. braunii assembly ................................................................... 38
4. Discussion ..................................................................................................................... 47
4.1 Performance of the binning method. ........................................................................ 47
4.2 Biology of the observed bacteria. ............................................................................. 48
4.2.1 Proteobacteria and Bacteroidetes. ............................................................................ 48
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4.2.2 Actinobacteria, Acidobacteria and Bacteroidetes. .................................................... 52
4.3 Origin of contamination ........................................................................................... 55
4.4 Future perspectives .................................................................................................. 56
5. Discussie ........................................................................................................................ 56
5.1 Beoordeling van de gebruikte methode. ................................................................... 57
5.2 Biologie van de waargenomen bacteriën .................................................................. 58
5.2.1 Proteobacteria en Bacteroidetes ............................................................................... 58
5.2.2 Actinobacteria, Acidobacteria en Planctomycetes. ................................................... 60
5.3 Toekomstperspectieven ........................................................................................... 61
6. Conclusion ..................................................................................................................... 63
7. Materials and methods .................................................................................................. 65
7.1 Sequencing data and assemblies. ............................................................................. 65
7.1.1 O. tauri and O. mediterraneus. ................................................................................. 65
7.1.2 P. crispa. ..................................................................................................................... 66
7.1.3 C. braunii. ................................................................................................................... 66
7.2 Preparation of the data prior to binning. .................................................................. 67
7.2.1 De novo assembly of non-algal contigs from O.tauri genome sequencing data using
CLC-assembly cell. ............................................................................................................... 67
7.2.2 Filtering of P. crispa assembly prior to binning. ........................................................ 68
7.2.3 Combining two German C. braunii draft assemblies using Newbler. ....................... 68
7.3 Binning of contigs with CONCOCT. ............................................................................ 69
7.6 Binning evaluation using taxonomic labels provided by MEGAN5. ............................ 69
7.7 Binning evaluation using single-copy core genes. ...................................................... 70
7.6 Isolation of bacterial genomes and scaffolding with Sspace. ..................................... 71
7.7 Aligning isolated genomes to reference using MUMmer. .......................................... 71
7.8 Evaluation of CONCOCT-assisted binning for separating prokaryotic and eukaryotic
sequences. ..................................................................................................................... 72
8. References ..................................................................................................................... 74
9. Addendum ..................................................................................................................... 92
9.1 Scripts ...................................................................................................................... 92
9.1.1 CONFPLOT.R ................................................................................................................. 92
9.1.2 CLUSTERPLOT.R .............................................................................................................. 92
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9.1.3 MEGAN_TO_CONCOCT.PY ................................................................................................ 94
9.1.4 MEGAN_CONCAT_TAXON.PY ............................................................................................ 94
9.1.5 CUT_FASTA.PY ............................................................................................................... 99
9.1.6 SCAFFOLD2CONTIGS.PL .................................................................................................. 100
9.1.7 COUNT_FRAGMENTS.SH ................................................................................................. 101
9.1.8 MFASTA_TOOLS.PL ....................................................................................................... 103
9.2 Supplementary figures ............................................................................................... 119

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Table of contents

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List of abbreviations
List of abbreviations
BCC Banyuls-sur-mer Culture Collection
BIC Bayesian Information Criterion
bp base pairs
CCALA Culture Collection of Autotrophic Organisms
CDD Conserved Domain Database
COG Clusters of Orthologous Group
DGGE Denaturing Gradient Gel Electrophoresis
ENA European Nucleotide Archive
Gbp Giga base pairs
kbp kilo base pairs
LCA Lowest Common Ancestor
Mbp Mega base pairs
NGS New Generation Sequencing
PCA Principal Component Analysis
PES Provasoli Enriched Seawater
RCC Roscoff Culture Collection
RPS-BLAST Reversed Position Specific BLAST
SCG Single Copy Core Gene
T-RFLP Terminal Restriction Fragment Length Polymorphisms (T-RFLP)

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List of abbreviations

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