La capture de la fonction par des approches haut débit

Transcription

1 Colloque Génomique Environnementale LYON 2011 La capture de la fonction par des approches haut débit Pierre PEYRET J. Denonfoux, N. Parisot, E. Dugat-Bony, C. Biderre-Petit, D. Boucher, G. Fonty, E. Peyretaillade Laboratory Microorganisms Genome and Environment UMR CNRS 6023 Clermont-Ferrand, FRANCE

2 Microorganisms Background Represent the most important and diverse group of organisms Widely distributed in many environmental habitats Play major roles in ecosystems functioning But Microbial diversity Diversity and structure of complex microbial communities still poorly understood Less than 1% of Bacteria cultivated and characterized (Amann et al., 1995) Great challenge in microbial ecology to evaluate microbial diversity in complex environments

3 Background Complex environments survey Access to a much larger reservoir of genomic and metabolic information Link community structure and diversity to function How? ATATGCGATA TATACGCTAT What? Who?

4 Genomic tools - Microbial species or microbial consortia characterization using nucleic acidbased techniques Global approaches DNA Soil sample : nucleic acids extraction RNA active cells PCR DNA fingerprints ARDRA TRFLP DGGE TGGE RAPD sequencing Genomic library Metagenomic Systematic sequencing Hybridization screening Functional screening Microarrays PCR Taxonomic - Functional genomic 4 RT-PCR sequencing

5 III. Genomic tools 11 Selective extraction of individual microorganisms from native microbial communities sample SIP : stable isotope probing Flow cytometry cell sorting (Kalyuzhnaya et al, 2006) Microfluidic chips (DNA, RNA isolation) (Marcy et al, 2007) Shotgun libraries Sequencing (Kalyuzhnaya et al, 2008) Whole genome amplication (MDA) Targeted metagenome : link between structure - function Whole genome amplication (MDA) Immunomagnetic capture (Pernthaler et al, 2008) Pyrosequencing

6 Metagenomics Culture-independent tool Enrich the knowledge and understanding of the uncultured world Increase of the rate of novel genes and novel molecule discovery Direct Sequencing Direct sequencing allowed by next generation sequencing (NGS) platforms development 454 pyrosequencing Difficult to apply for a complete survey of complex environmental samples A promising alternative would be to reduce sample complexity Metagenomics by targeted capture methods

7 CH 4 Production MCRI et MCRII ANME (anaerobic methane-oxidizing archaea)

8 Lake Pavin [CH 4 ] [CH 4 ] [CH 4 ] Mixolimnion (0-60m) [CH 4 ] Chemocline Monimolimnion (63-92m) [Fe 2+ ] [CH 4 ] [CH 4 ] [NH 4+ ] [CH 4 ] Zone intermédiaire Sédiments

9 Method validation Targeting mcra gene on Methanosarcina acetivorans C2A strain qpcr analysis and enrichment calculation Based on the R=2 - Ct calculation (Livak and Schmittgen, 2001) Relative fold enrichment (R) 1 st cycle nd >200,000 cycle Tremendous increase of mcra enrichment with two cycles of capture Sequencing of DNA fragments retrieved from the second cycle mcra (1713 bp) M. acetivorans C2A 1800bp High potential to target desired sequence and its flanking region All sequences showed a perfect correspondance to the mcra gene Possibility to capture a 1800 bp sequence with a 200bp non-coding region

10 Different Strategies of Sequencing Lake Pavin 90m Metagenomic DNA Sample Targeted Gene (mcra) Amplicons (PCR) Metagenome Targeted Capture

11 Preprocessing Extract Data Split by Multiplex Identifier (MID) Extract Sequence and Quality Data Trim Ends Trim 5 (Key/MID/PCR Primer) and 3 (Adaptator B) Ends Trim Low Quality Ends Filter Sequences Filter Too Short and Too Long Reads (Mode ± 2SD) Filter Low Quality Reads Filter Reads with Ambiguous Bases N Filter Low Complexity Reads Filter Artificial Duplicates

12 >Seq1 ATGCACGTAGCT ACGAAATCA >Seq2 ATCGACTCAGAC AGCCACGAC >Seq3 ATCGACGACTCA GACCACAC.. >Seq1 ATGCACGTAGCT ACGAAATCA >Seq2 ATCGACTCAGAC AGCCACGAC >Seq3 ATCGACGACTCA GACCACAC.. >Seq1 ATGCACGTAGCT ACGAAATCA >Seq2 ATCGACTCAGAC AGCCACGAC >Seq3 ATCGACGACTCA GACCACAC.. mcra Sequences Database Find mcra sequences >Seq1 FTQYEAAALVAAR RDEAAL >Seq2 FTQYEAAALVAGR RDEAAL >Seq3 FTQYEAAALVGAR RDEAAL.. >Seq1034 FTQYEAAALVAAR RDEAAL >Seq5532 FTQYEGAALVALA RDEAW.. >Seq41 FTQYEAAALVAAR RDEAAL >Seq65 AAALVAARRDEAA LGLKDEAA >Seq193 FTQYEGAALVALA RDEAW.. Amplicons Metagenome Capture

13 Diversity >Seq1034 FTQYEAAALVAAR RDEAAL >Seq5532 FTQYEGAALVALA RDEAW.. Metagenome >Seq1 FTQYEAAALVAAR RDEAAL >Seq2 FTQYEAAALVAGR RDEAAL >Seq3 FTQYEAAALVGAR RDEAAL.. Amplicons Capture >Seq41 FTQYEAAALVAAR RDEAAL >Seq65 AAALVAARRDEAA LGLKDEAA >Seq193 FTQYEGAALVALA RDEAW..

14 Assembly Overlap Length : 60 nts Percent Identity Overlap : 95% Reads Assembled Singletons Contigs Large Contigs Average Contig Size N50 Contig Size Largest Contig Size Metagenome Capture mcr mcrb mcrc mcrd mcrg mcra Contigs

15 Diversity Strategy OTUs Amplicons 28 Capture 24 Metagenome 1 Published data 3

16 Percentage of Reads Diversity Capture Amplicons OTUs

17 Diversity Number of OTUs Amplicons Metagenome Capture Methanopyrales Methanobacteriales Methanococcales Novel Order Methanomicrobiales Methanocellales Methanosarcinales

18 Diversity 100% Highest diversity retrieved by targeted capture strategy Detection of a deeper diversity in Methanosarcinales and Methanobacteriales orders compared to a PCR based approach 90% 80% 70% 60% 50% 40% 30% Methanomicrobiales Methanosarcinales Methanobacteriales Novel Order 20% 10% 0% Capture Amplicons

19 Environmental application Targeting large mcra gene diversity on Lake Pavin (France) Background (3/3) Solution hybrid selection method Method validation Env. application 70m CH 4 90m Mixolimnion (oxic zone) Mesolimnion (interface) Monimolimnion (anoxic zone) Conclusions & Sediments 92m Perspectives Significant mcra enrichment and ability to recover large DNA fragments than PCR approach

20 Conclusions & Perspectives Background (3/3) Solution hybrid selection method Method validation Env. application Conclusion s & Perspective s Innovative strategy to trap desired targets in complex mixtures Recovery of larger DNA fragments than PCR approach Relevant method to enrich in targeted sequences Ability to use a large and explorative probe set Target simultaneous phylogenetic and functional markers Capture largest DNA fragments Complementary and beneficial approach to NGS Suitable tool to investigate genetic diversity in complex environments

21 Laboratory Microorganisms: Genome and Environment Acknowledgments GIIM team P. Peyret J. Denonfoux E. Dugat-Bony N. Parisot F. Jaziri S. ComtetC. Biderre-Petit D. Boucher S. Rimour E. Peyretaillade A. Moné B. Chebance I. Pinto Thank you for your attention! PhD Funder CNRS Région Auvergne