Deep sequencing to understand the marine environment. Jan Pawlowski Department of Genetics and Evolution University of Geneva

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1 Deep sequencing to understand the marine environment Jan Pawlowski Department of Genetics and Evolution University of Geneva

2 Known 1/10 gap In the Ocean the number of predicted species is about 10 times higher than the number of catalogued species (200,000). (Mora et al. How many species are there on Earth and in the Ocean PLoS Biol 2011) How big will be this gap if we add meiofauna (animals < 0.5 mm), protists, and bacteria? Unknown 1/100 gap?

3 Meio- and microeukaryotes the key players in marine ecosystem Marine protists and small-size animals play a crucial role in ecosystem functioning, however, their diversity is highly underappreciated because they are small, morphologically indistinctive and usually uncultivable.

4 The scarcity of marine eukaryotes genomes Despite their ecological and evolutionary importance, the complete genome data are available for very few marine microbial eukaryotes: Perkinsus marinus Phaeodactylum tricornutum Thalassiosira pseudonana Aureococcus anophagefferens Ectocarpus siliculosus Bigellowiella natans Emiliania huxleyi Micromonas Ostreococcus Guillardia theta Monosiga brevicollis Burki et al. 2012

5 The abundance of unknown genes About 80% of unclassified sequences in the transcriptomes of two major groups of marine protists Hits against Genbank nt database (R. Sierra, unpubl.) Forams Radiolarians

6 Next generation sequencing (NGS) is changing our view of marine eukaryotes diversity DNA extraction Sequencing of selected taxa Environmental sample DNA of all species present in the environment Next generation sequencing (NGS) of global diversity Metagenetics (metabarcoding) Species richness

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8 Tara-Oceans 1 st Metabarcoding campaign: 35 stations 260 discrete samples various depths and size fractions Total of 426,885,078 ILLUMINA sequences, corresponding to 9,074,257 distinct sequences each sequence is present 47 times in average de Vargas 2012

9 NGS reveals the extraordinary diversity of marine microeukaryotes Catalogued species A. (Ntot 2 million) B. Environmental OTUs ( S V4 rdna 97%) Metazoa Fungi Streptophyta Stramenopila Alveolata Archaeplastida Rhizaria Amoebozoa Excavata Hacrobia Opisthokonta PROTISTS

10 INCERTA Breviatea Mycetozoa Variosea Discosea Lobosa incertae sedis Tubulinea + Arcellinida Euglenozoa Heterolobosea Jakobida Fornicata Parabasalia Preaxostyla Malawimonadidae Choanoflagellida Mesomycetozoa Chlorophyta Glaucocystophyta Rhodophyta Centroheliozoa Cryptophyta Haptophyta Katablepharidophyta Picobiliphyta Telonemia Apicomplexa Chromera Ciliophora Dinophyceae Ellobiopsidae Perkinsea Syndiniales Bacillariophyta Bicoecea Bolidophyceae and relatives Chrysophyceae-Synurophyceae Dictyochophyceae Eustigmatophyceae Hyphochytriomyceta Labyrinthulea MAST Oomyceta Opalinata Pelagophyceae Phaeophyceae Phaeothamniophyceae Picophagea Pinguiophyceae Pirsonia Raphidophyceae Xanthophyceae Acantharea Polycystinea RAD A RAD B RAD C Cercozoa Foraminifera Apusomonadidae Hilomonadea 1 million ~580,000 taxonomically assigned OTU_97% 100,000 10,000 1, AMOEBOZOA EXCAVATA HACROBIA ALVEOLATA RHIZARIA STRAMENOPILA Metazoa: 2 million sp / 226,000 OTUs Fungi: 380,000 sp / ~4000 OTUs Streptophyta: 350,000 sp / 160 OTUs

11 NGS challenges classical taxonomic knowledge Lecroq et al. PNAS, 2011

12 NGS demonstrates the limits of current genetic databases 100% 90% 80% 70% 60% 50% 40% 30% 20% genus > 95% < 95% 10% 0% DSE1 (1636) DSE2 (1539) DSE3 (1218) DSE4 (918) DSE5 (1226) DSE6 (1778) Percentage of 18S V9 OTUs from 454-based study of deep-sea floor showing > 95% similarity to the GenBank sequences and assigned to the genus level (data from Pawlowski et al., PLoS One 2011)

13 NGS data requires careful analysis artifactual polymorphism chimeras taxonomic assignment 16/11/10

14 NGS data interpretation demands substantial scaling up of existing databases 1. Multigene database to identify new lineages and establish their phylogenetic relationships 2. DNA barcodes library to identify species using short DNA fragments 3. Microscopic documentation of organisms belonging to lineages known only by their sequences (reverse taxonomy) FISH detection of unknown foraminiferal lineages (L.Perret-Gentll, unpubl)

15 NGS future applications How NGS can help increasing the productivity and sustainability of marine environment? Biomonitoring of sites impacted by human activities (aquaculture, offshore drilling, deep seabed mining) Bioprospection search for biological resources for commercial and industrial exploitation Understanding of global ecosystem functioning Reconstruction of past environmental changes

16 An example: NGS-based deep seabed monitoring The deep-sea floor is vulnerable to anthropogenic activities and particularly suitable for NGS-based biomonitoring, because: the diversity is very high the species are rare their identification is difficult their cultivation is usually impossible

17 Conclusions Deep sequencing offers an exceptional tool for exploring and surveying the marine environment. However, to make it working we need: funding for developing genomic and barcoding databases public-private partnership to test application of deep sequencing in ecological analyses development of standards based on deepsequencing data