Next Generation Sequencing Technologies in Microbial Ecology. Frank Oliver Glöckner

Transcription

1 Next Generation Sequencing Technologies in Microbial Ecology Frank Oliver Glöckner 1

2 Max Planck Institute for Marine Microbiology Investigation of the role, diversity and features of microorganisms Interactions with physical and chemical processes in marine and other aquatic habitats Founded 1992 in Bremen, Germany 2

3 Marine Microbiology at MPI a Holistic Approach Who is out there and How much of which kind? What are they doing and Under which conditions are they doing what? 3

4 Promise of NGS: Much Denser Network of Data Phylogenetic diversity Qualitative data Quantitative data Organisms Environment Functional diversity Functional inventory Operon structures Expression profiles x, y, z, t Environmental descriptors -> Integrated datasets Genes 4

5 Data Integration Kottmann et al., NAR, submitted 5

6 Ecological Genomics The Vision Statistics Key parameters Ecosystems Biology Modelling Predictions 6

7 Ribosomal RNA as a universal marker gene Full cycle rrna-approach sample extracted nucleic acids DNA rrna nucleic acid probe rdna clones Pyrosequencing rdna sequences comparative analysis rdna dataset Amann, 1995 hybridization phylogeny 7

8 Diversity Analysis Sample Clone lib month PCR High diversity Pedros-Alio, Trends in Microbiology, 2006, vol. 12, issue 6, page 257 8

9 Diversity Analysis Sample Clone lib month PCR High diversity Tags 10,000-50,000 1 week 9

10 Problems Processing the data Accuracy/Quantitative? DNA/RNA extraction Multiple Operons Technical replicates Noise (sequencing errors ) 10

11 SILVA Databases Specifications Comprehensive & Aligned Bacteria, Archaea, Eukarya SSU, LSU Regularly updated Quality first Quality management Transparent process documentation Integrative Nomenclature Taxonomy Cultured, Typestrains Habitat (r100) 11

12 Growth of rrna databases (RDP & SILVA) Growth of SSU ribosomal RNA databases (RDP II & SILVA) rrna Sequences Comprehensive ribosomal RNA databases Pruesse et al. NAR 2007, vol. 35, no 21, page SILVA

13 SILVA SSURef 100: Fully classified guide tree 13

14 ARB Software Suite A Software Environment for Sequence Data Ludwig et al. NAR, 2004 ARB 5.0, 64 bit version released on 04. September

15 Problems Processing the data Accuracy/Quantitative? DNA/RNA extraction Multiple Operons Noise (sequencing errors ) Technical replicates 15

16 Accuracy The rare biosphere : a reality check Reeder and Knight, 2009, Nature Methods vol. 6, no. 9, p

17 SILVA SSUParc 100, Sequence Length Distribution Comprehensive ribosomal RNA databases rrna Sequences Length (bases) 17

18 Technical Replicates I Helgoland Sample , 454 Ti 1/2 PTP Gomez-Alvarez et al., 2009, ISME Journal, pages

19 Technical Replicates II Helgoland Sample , 454 Ti 1/2 PTP 19

20 Unclassified Viridiplantae TA06 Spirochaetes SHA-109 Rhodophyta et al. Planctomycetes ML635J-21 Metazoa Lentisphaerae Gemmatimonadetes Gammaproteobacteria_4 20 Gammaproteobacteria_3 Gammaproteobacteria_2 Gammaproteobacteria_1 Gammaproteobacteria Fusobacteria Technical Replicates III - Dereplication 25,0% SSU Dereplicated 20,0% 15,0% 10,0% 5,0% 0,0% Fungi Firmicutes Euryarchaeota Euglenozoa Epsilonproteobacteria Deltaproteobacteria Deferribacteres Cyanobacteria Crenarchaeota Chloroflexi Chlorobi Candidate division WS6 Candidate division WS3 Candidate division WS1 Candidate division TM6 Candidate division SR1 Candidate division OP8 Candidate division OP3 Candidate division OP11 Candidate division OD1 Candidate division BRC1 Betaproteobacteria BD1-5 Bacteroidetes Amoeba Actinobacteria Acidobacteria

21 A Bioinformatic Workbench for Ecological Genomics Comprehensive ribosomal RNA databases A Software Environment for Sequence Data Organisms Environment Genes 21

22 Functional Diversity Analysis Sample Fosmids Random sequencing ~ ORFs NGS ,000 ORFs DNA 40, month End sequencing 20,000 ORFs High diversity 22

23 Fosmid Sequencing Fosmids: 42 PTP: 1/4, 454 Ti min.: 101 bp max.: 48,265 bp mean: 8,085 bp Costs: 3000 Euro 48 contigs > 10 kb 23

24 Functional Diversity Analysis Sample Tags DNA 500,000-1 Mio 1 week Statistics BLAST COGs Classify Assembly? High diversity 24

25 Assembly 25

26 Are we prepared for the Data Flood? 26

27 Computing Infrastructure Cooling - Liquid cooling with 5,000 L/h at 8 C Power kw at peak - installed: 40 kva Storage - 8 TByte RAID file server - 4 TByte RAID database server Computers -43 cluster nodes - several larger servers -400 CPU cores 27

28 Moore s Law - Outcompeted 28

29 Take Home Message for the Next Generation Biologists Three languages! Mother tongue English Perl, Phyton Data management Garbage in -> garbage out! Standardisation 29

30 The Group Thanks for your attention 30