1. 1. Annotating 18S rDNA sequences from environmental molecular surveys Ramon Massana EukRef Workshop, Vancouver, Canada, 21 July 2015
2. 2. Microorganisms in the marine plankton 1977 - Many bacteria Hobbie et al. 1977. Appl. Environ. Microbiol. 1980 - Very active Fuhrman & Azam 1980. Appl. Environ. Microbiol. 1982 - Controlled by predation by small flagellates Fenchel 1982. Mar. Ecol. Progr. Ser. The microbial loop Phytoplankton Half Earth primary production occurs at the sea Field et al. 1998. Science Marine primary producers are essentially planktonic microorganisms At the base of food webs Classical food web Microbial loop Azam et al. 1983. Mar. Ecol. Progr. Ser Chlorophyll concentration by SeaWiFS September 1997 August 2000
3. 3. A simple question: Who are the smallest marine protists? Morphology Cultures Molecular Nanoplankton (2-20 m)Microplankton (20-200 m) Picoplankton (0.2-2 m) 1000-10,000 cells ml-1 in seawater
4. 4. Advantages Universal in all living beings Mosaic of conserved and variable regions Highly expressed (DNA and RNA approaches) The 18S rDNA gene as a phylogenetic marker Limitations Variable rDNA operon copy number Different evolutionary rate among lineages Primers used affect the results
5. 5. Novel lineages were very apparent in molecular surveys of marine picoeukaryotes Mainly heterotrophs Mainly phototrophs Novel alveolates Novel stramenopiles Blanes Bay Dez et al. 2001 Moon van der Staay et al. 2001 Antarctica Mediterranean seaNorth Atlantic Equatorial Pacific Massana et al. 2004
6. 6. Oomycetes Hyphochytrids Blastocystis Labyrinthulids Thraustochytrids Phototrophic stramenopiles Placididea Pirsonids MAST-1 Bicosoecids Developayella MAST-2 MAST-3 MAST-12 MAST-7,-8 MAST-4 MAST-11 MAST-6,-9,-10 Massana et al. 2004 MAST lineages (Marine Stramenopiles) 34 complete 18S rDNA sequences 153 partial sequences
7. 7. Identifying MAST cells MAST-1B MAST-1C 10 m MAST-4 Massana et al. 2006 MAST-4MAST-1CMAST-1B Important bacterial grazers in the sea ! FISH Abundant an widely distributed ! Small (2-5 m) free-living unpigmented protists
8. 8. Objectives Evaluate the consistency of the groups described so far Explore the existence of other groups Describe substructure within the groups
9. 9. MAST criteria Belong to the basal heterotrophic stramenopiles Do not belong to any described group in this region
10. 10. 18 MAST clades
11. 11. 0.05 FJ431722_PLA_OXIC_ATL HQ868602_PLA_OXIC_PAC HQ868974_PLA_OXIC_PAC HQ868964_PLA_OXIC_PAC HQ222553_PLA_OXIC_POL HQ868790_PLA_OXIC_PAC HQ868707_PLA_OXIC_PAC HQ869132_PLA_OXIC_PAC HM369550_PLA_OXIC_ATL HQ869095_PLA_OXIC_PAC GU823321_PLA_OXIC_ATL HQ869551_PLA_MICRO_PAC HQ868750_PLA_OXIC_PAC HQ867292_PLA_OXIC_PAC HM369537_PLA_OXIC_ATL HQ868869_PLA_OXIC_PAC HQ868556_PLA_OXIC_PAC GU823363_PLA_OXIC_ATL EU500143_MAST10 AA538_E14_SAG HQ870429_PLA_OXIC_PAC HQ870293_PLA_OXIC_PAC HQ868825_PLA_OXIC_PAC HQ868512_PLA_OXIC_PAC HQ868743_PLA_OXIC_PAC HQ869229_PLA_OXIC_PAC GQ344735_PLA_OXIC_MED JN693076_PLA_OXIC_PAC GU823606_PLA_ANOX_ATL HQ869079_PLA_OXIC_PAC GQ344707_PLA_OXIC_MED JQ955868_PLA_OXIC_POL AB242_B02_SAG HQ870138_PLA_OXIC_PAC HQ868615_PLA_OXIC_PAC JQ782091_PLA_OXIC_PAC AA539_D16_SAG HQ868708_PLA_OXIC_PAC HQ868837_PLA_OXIC_PAC HQ868543_PLA_OXIC_PAC HM561229_PLA_OXIC_POL HM561230_PLA_OXIC_POL AF363208_PLA_OXIC_POL HQ868770_PLA_OXIC_PAC HQ868710_PLA_OXIC_PAC GU823331_MAST10 HQ868739_PLA_OXIC_PAC HQ869097_PLA_OXIC_PAC FJ971801_PLA_OXIC_POL HQ868631_PLA_OXIC_PAC GU823153_PLA_MICRO_ATL AA538_L13_SAG JQ782009_PLA_OXIC_PAC GU823326_PLA_OXIC_ATL GU823095_PLA_OXIC_ATL EU561940_PLA_OXIC_IND HQ867665_PLA_OXIC_PAC HQ865050_PLA_OXIC_PAC HQ869384_PLA_OXIC_PAC HQ868624_PLA_OXIC_PAC GU823098_PLA_OXIC_ATL HQ868864_PLA_OXIC_PAC HM561231_PLA_OXIC_POL AY116220_PLA_OXIC_POL HQ868504_PLA_OXIC_PAC 0 0 0 9 1 3 0 100 0 0 9 3 0 9 0 100 0 100 9 9 0 0 100 9 3 0 0 0 8 5 0 6 2 100 0 0 0 2 4 100 0 0 100 100 0 100 0 9 7 0 0 0 0 0 0 0 0 7 100 0 0 6 5 6 6 0 100 100 0 0 9 3 9 3 7 5 Analyzing the substructure MAST- 8
12. 12. 1 Download as much sequences as possible (and then discard) Tips in making the final reference dataset 2 Rely on Sanger sequencing Very clear perception of quality Multiple sequencing reactions from the same amplicon => complete 18S rDNA sequences
13. 13. 1 Download as much sequences as possible (and then discard) Tips in making the final reference dataset 2 Rely on Sanger sequencing 0 10 20 30 40 50 1 51 101 151 201 251 301 351 401 0 10 20 30 40 50 1 51 101 151 201 251 301 351 401 451 But No chromatograms Apply a quality criteria AND check !! HTS Faster, easier cheaper and more Automatic pipelines
14. 14. 1 Download as much sequences as possible (and then discard) Tips in making the final reference dataset 2 Rely on Sanger sequencing 3 Make phylogenies Verify the annotation of sequences to given groups Be suspicious of long orphan branches (individual BLAST)
15. 15. 1 Download as much sequences as possible (and then discard) Tips in making the final reference dataset 2 Rely on Sanger sequencing 3 Make phylogenies 4 Be aware that even Sanger can have sequencing errors Ends need to be correctly trimmed In databases you do not see the chromatograms
16. 16. 1 Download as much sequences as possible (and then discard) Tips in making the final reference dataset 2 Rely on Sanger sequencing 3 Make phylogenies 4 Be aware that even Sanger can have sequencing errors 5 Chimeras do occur, can be very frequent, and can escape detection algorithms
17. 17. Stramenopile diversity. The importance of quality and chimera checks Chimeras appearing within MAST-4 0.03 Backbone tree Seed to align 454 reads
18. 18. Stramenopile diversity. The importance of quality and chimera checks 0.02 chimera.slayer (Mothur) Removes 81 chimeras 0.03 0.02 Manual checking Removes 6 chimeras Chimeras appearing within MAST-4
19. 19. 1 Download as much sequences as possible (and then discard) Tips in making the final reference dataset 2 Rely on Sanger sequencing 3 Make phylogenies 4 Be aware that even Sanger can have sequencing errors 5 Chimeras do occur, can be very frequent, and can escape detection algorithms Since the 18D rDNA is such a conserved gene Be very conservative to accept a new phylotype!!