COMMUNITY STRUCTURE OF BENTHIC BACTERIA OF THE

HUMBOLDT CURRENT ECOSYSTEMV. A. GALLARDO, C. ESPINOZA, A. FONSECA

Departamento de OceanografíaUniversidad de Concepción

Census of Marine Life (CoML) / Sloan FoundationInternational Census of Marine Microbes (ICoMM) & Keck Foundation)

PROGRAM: “Microbial Population Structure of the World’s Oceans”

Cape Town, June - 2009

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.

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.

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.

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.

ICoMM´S:“UNVEILING THE OCEAN’S HIDDEN MAJORITY

THROUGH 454 TAG SEQUENCING”

• Apr. 2007 – Call for proposals• Jun. 2007 –Presentation of proposals• Nov. 2007 – Our proposal highly rated, approved

for 16 samples (42 projects in total)• Dec. 2007 – First sampling.• Dec. 2008 – Fourth and last sampling.• Mar. 2009 - Full database available in VAMPS.

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.

SAMPLING STATIONS

“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.

m

TOTAL NROFSEQS =351,751

Revised: April 2nd 2009

SPRING

SPRING

SUMMER

WINTER

40 PHYLA (294,839 tag seqs., 95%)

TOTAL326,344 tag seqs.

% PHYLA

TOTAL326,344 tag seqs.

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

% CLASSES

TOTAL326,344 tag seqs.

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

232 FAMILIES (147,631 tag seqs., 45%)

TOTAL326,344 tag seqs.

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

% DOMINANCE

RAREFACTION - STA. 1 (15m)Rarefaction Plot - Phyla - ST 1

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RAREFACTION – STA. 4 (27m)Rarefaction Plot - Phyla - ST 4

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Rarefaction Plot - Classes - ST 4

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RAREFACTION – STA. 7 (35m)Rarefaction Plot - Phyla - ST 7

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RAREFACTION- STA. 18 (88m)

Our undescribed as yet spirochaete

FILMED BY THOMAS ISHOEY AT SYNTHETIC GENOMICS, 2008

BACTERIAL MAT

EDITION: CAROLA ESPINOZA 2008

HYPOXIC / ANOXIC ZONES

DEAD ZONES?

THANKS