Annotating 18S rDNA sequences from environmental molecular surveys Ramon Massana EukRef Workshop, Vancouver, Canada, 21 July 2015
Transcript
1. Annotating 18S rDNA sequences from environmental molecular
surveys Ramon Massana EukRef Workshop, Vancouver, Canada, 21 July
2015
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. 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. 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. 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
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. Objectives Evaluate the consistency of the groups described
so far Explore the existence of other groups Describe substructure
within the groups
9. MAST criteria Belong to the basal heterotrophic
stramenopiles Do not belong to any described group in this
region
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. 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. 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. 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. 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. Stramenopile diversity. The importance of quality and
chimera checks Chimeras appearing within MAST-4 0.03 Backbone tree
Seed to align 454 reads
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. 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!!