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

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

% PHYLA TOTAL 326,344 tag seqs.

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

% CLASSES TOTAL 326,344 tag seqs.

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

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

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

% DOMINANCE

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

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

RAREFACTION – STA. 7 (35m)

RAREFACTION- STA. 18 (88m)

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

EDITION: CAROLA ESPINOZA 2008 BACTERIAL MAT EDITION: CAROLA ESPINOZA 2008

HYPOXIC / ANOXIC ZONES DEAD ZONES?

THANKS