First EukRef Workshop (Vancouver 2015)

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 µm)Microplankton (20-200 µm) Picoplankton (0.2-2 µm)
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
Díez et al. 2001
Moon van der Staay et al. 2001
Antarctica
Mediterranean seaNorth Atlantic
Equatorial Pacific
Massana et al. 2004

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. Identifying MAST cells
MAST-1B MAST-1C
10 µm
MAST-4
Massana et al. 2006
MAST-4MAST-1CMAST-1B
Important bacterial grazers in the sea !
FISH
Abundant an widely distributed !
Small (2-5 µm) 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

10. 18 MAST clades

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

13. 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

14. 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

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
Verify the annotation of sequences to given groups
Be suspicious of long “orphan” branches (individual BLAST)

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
Ends need to be correctly trimmed
In databases you do not see the chromatograms

17. 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

18. Stramenopile diversity. The importance of quality and chimera checks
Chimeras appearing within MAST-4
0.03
Backbone tree
Seed to align 454 reads

19. 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

20. 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!!