Single-cell genomics applied to the picobiliphytes using next-generation sequencing

Transcription

1 Department of Ecology, Evolution and Natural Resources and Institute of Marine and Coastal Sciences Rutgers University, NJ Single-cell genomics applied to the picobiliphytes using next-generation sequencing Dana Price 1, Debashish Bhattacharya 1, and Hwan Su Yoon 2 1 Bhattacharya Lab, Rutgers University 2 Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine, USA

2 The Picobiliphyta F. Not et al., Science 315, 253 (2007)

3 The Picobiliphyta nucleus ssrna nucleomorph PBP organelle Recently described lineage of pigmented eukaryotes Identified via 18S rdna within ocean waters Autofluorescence suggests phycobilin proteins DAPI staining reveals a nucleomorph Photosynthetic lifestyle via secondary endosymbiosis

4 The Picobiliphyta Cosmopolitan (Arctic Ocean, Mediterranean, Barnegat NJ) Habitats extremely varied (4 89m depth, 5-30 C) Placed as sister to Cryptophytes and Katablepharids

5 The Picobiliphyta Ocean metagenomic analyses at Boothbay Harbor, ME consistently recovered picobiliphytes in the heterotrophic (Lysotracker) FACS sorted fraction None sorted via chlorophyll autoflourescence

6 Sampling 50 ml seawater collected from dock, Boothbay Harbor, ME

7 FACS Sorting Single cells sorted through 10 μm filter via Lysotracker, lack of chlorophyll autoflourescence

8 Lysis/Amplication Individual cells lysed, subject to whole-genome amplification (WGA/MDA)

9 Identification via rdna 454 sequencing ca. 90Mbp each 6/35 genomes were Picobiliphytes

10 Assembly 4-5.3Mbp unassembled

11 Virus Novel 1804bp ssdna virus Largest characterized nanovirus Rep protein, second opposite-sense ORF Nested within GOS sequence data

12 Virus Even read coverage across genome suggests infection * Our methods cannot rule out viral attachment

13 BLASTx analysis of total genome data revealed no photosystem proteins

14 Illumina genomic DNA libraries constructed for MS and MS Generated 12Gbp total between the two cells Co-assembled with 454 data

15 phylosort MS * A. Moustafa et al., Science 324, 1724 (2009) ~72,000 contigs 27 Mbp 5,231 trees BLASTX no photosystem proteins no photosystem proteins

16 What are they eating? Back to the contaminants Marine Bacteroidetes Proteobacteria Phage Larger dsdna viruses

17 Genome Size BLASTx of assembled contigs to proteins in other model eukaryotes: 32% of Saccharomyces cerevisae 30% of Phaeodactylum tricornutum BLASTx of assembled contigs to total database identified ~7.9 Mbp as eukaryotic Other pico-eukaryote genomes ~12 22Mbp in size If we assume 30-50% of genome is Picobiliphyte specific, ~ 50% of genome identified

18 Tree of Life Coassembly recovered many eukaryotic genes for which we had phylogenetic markers 2594 AA comprising the nuclear proteins actin, alpha-tubulin, beta-tubulin, HSP90, cytosolic HSP70, ribosomal pregulatory rotein L3, and 26S proteasome non-atpase subunit.

19 Tree of Life

20 Major Results from the first single-cell draft genome analysis Representatives of the three most divergent Picobiliphyte clades were sympatric in 50mL of sea water. In contrast to the recent description of the phylum, we find no evidence of plastid DNA or nuclear genes related to photosynthesis, suggesting these, and likely all Picobiliphytes are heterotrophs. Organelles described in the Not el al. (2007) paper were likely prey or temporary plastids (i.e., kleptoplasts). A significant fraction of DNA sequences from the second and third cells are related to Bacteroidetes and associated phages, suggesting these protists grazed phage-infected bacteria or large DNA viruses. Genome reads from MS584-5 are dominated by a novel single-stranded DNA nanovirus, possibly representing host cell infection. Phylogenomic affinity to Stramenopiles and other Chromalveolates leaves the door open for photosynthetic ancestry Culture independent techniques have implications from biomedical to biodiversity, provided we can apply proper technical and bioinformatic analyses

21 More info Science 332, 714 (2011)

22 Acknowledgements EF DEB OCE OCE

23 Questions

24 Danke.