1. Census of Marine Life (CoML) / Sloan Foundation
International Census of Marine Microbes (ICoMM) & Keck Foundation)
PROGRAM: “Microbial Population Structure of the World’s Oceans”
COMMUNITY STRUCTURE OF BENTHIC BACTERIA OF THE HUMBOLDT CURRENT ECOSYSTEM
V. A. GALLARDO, C. ESPINOZA, A. FONSECA
Departamento de Oceanografía
Universidad de Concepción
Cape Town, June

2. ICoMM´S PROGRAM: “MICROBIAL POPULATION STRUCTURE OF THE WORLD’S OCEANS”
With funding from the W.M. Keck Foundation, ICoMM established a facility for generating V-6 tag rRNA gene sequences from Bacteria and Archaea at the Marine Biological Laboratory in Woods Hole, MA, USA.
Adopted a massively-parallel, 454-based tag sequencing strategy that allows extensive study of marine microbial communities (not only of dominant forms).
This strategy is based on sequencing of hypervariable regions of the SSU rRNA gene (in our case V6).
Allows to measure both abundance and taxa richness
Allows efficient comparisons of their composition and structure.

3. METHODOLOGY
The Genome Sequencer™ GS20 System from 454 Life Sciences™ and Roche Applied Science is used.
It is a sequencing platform suitable for a wide range of applications, in this particular case for small RNA analysis and amplicon sequencing.
The Genome Sequencer FLX is built upon 454 Sequencing™ technology that allows 200–300 bp read lengths and very high single-read accuracy.
One application in which the technology is accelerating the field’s understanding is metagenomics.

4. METHODOLOGY (cont.)
The GS20 system uses DNA-capture beads that catch on average one short single-stranded template
Beads are distributed on a solid-phase sequencing substrate (a PicoTiterPlateTM) with 1.6 million wells.
Each well can hold a bead and additional reagents, including polymerase, luciferase, and ATP sulfurylase.
The template is then copied millions of times in an oil emulsion PCR (emPCR).
Microfluidics cycle each of the four nucleotide triphosphates over the PicoTiterPlateTM.
Incorporation of a nucleotide releases pyrophosphate, the substrate for a luminescence reaction which is recorded with a cooled CCD camera.
The record of intensity of each flow of a nucleotide is a flow-gram analogous to a chromatogram that reports the order of A, C, G and T residues from a DNA sequencing template.

5. THE GENOME SEQUENCER FLX GS20
Supports a number of formats allowing users to customize the nr. of samples /instrument run and the nr. of reads/sample.
A single run can be physically divided into 2, 4 or 16 samples with 210,000 reads per sample,
70,000 reads per sample and 12,000 reads per sample.

6. ICoMM´S: “UNVEILING THE OCEAN’S HIDDEN MAJORITY THROUGH 454 TAG SEQUENCING”
Apr – Call for proposals
Jun –Presentation of proposals
Nov – Our proposal highly rated, approved for 16 samples (42 projects in total)
Dec – First sampling.
Dec – Fourth and last sampling.
Mar Full database available in VAMPS.

7. HUMBOLDT PROPOSAL
Proposal to apply the 454 tag sequencing technology to sediment microbial communities under the influence of the oxygen minimum zone of an Eastern Boundary Current ecosystem: the Humboldt marine ecosystem.

8. SAMPLING STATIONS

9. “SPECIES” OF BACTERIA
The method treats each V6-rRNA tag as a proxy for the presence of an organism (whatever taxon is warranted), i.e., species, genus, family, order, class or phylum:
Allows the identification of the different kinds of taxa and their abundance in a community.

11. m TOTAL NR OF SPRING SEQS = 351,751 SUMMER WINTER SPRING
Revised: April 2nd 2009 m
TOTAL NR OF
SEQS =
351,751
SPRING
SUMMER
WINTER
SPRING

14. 40 PHYLA (294,839 tag seqs., 95%)
TOTAL
326,344 tag seqs.

15. % PHYLA
TOTAL
326,344 tag seqs.

16. 42 CLASSES (228,784 tag seqs., 70%)

17. % CLASSES
TOTAL
326,344 tag seqs.

18. 89 ORDERS (185,002 tag seqs, 58%)

19. 232 FAMILIES (147,631 tag seqs., 45%)
TOTAL
326,344 tag seqs.

20. 671 GENERA (68,610 tag seqs., 21%)

21. % DOMINANCE

22. RAREFACTION - STA. 1 (15m)
PHYLA
CLASSES
ORDERS
FAMILIES
GENERA

23. RAREFACTION – STA. 4 (27m)
PHYLA
CLASSES
ORDERS
FAMILIES
GENERA

24. RAREFACTION – STA. 7 (35m)

25. RAREFACTION- STA. 18 (88m)

31. Our undescribed as yet spirochaete
FILMED BY THOMAS ISHOEY AT SYNTHETIC GENOMICS, 2008

32. EDITION: CAROLA ESPINOZA 2008
BACTERIAL MAT
EDITION: CAROLA ESPINOZA 2008

33. HYPOXIC / ANOXIC ZONES
DEAD ZONES?

34. THANKS