Sequencing All of Microbial Life: Challenges and Opportunities Rob Edwards Argonne National Laboratory San Diego State University

First bacterial genome 100 bacterial genomes 1,000 bacterial genomes Number of known sequences Year How much has been sequenced Environmental sequencing

Everybody in Toronto Everybody in North America All cultured Bacteria 100 people How much will be sequenced One genome from every species Most major microbial environments

Rank Abundance Curves, Papers vs Genomes Microbial publications vs Genomes by Family

16S Abundance -- Human Intestine

16S Abundance -- Upland Pasture Soil

Environmental Genomics -- Wisconsin Soil

Line Island Metagenomics Transect

Environmental Genomics -- Whale Fall

There are big gaps in sequence space 6,400 total taxa About 380 are human, animal or plant pathogens 360 complete prokaryotic genomes published 56 archaeal and 940 bacterial genomes in progress –~400 are pathogens Approximately ~5,000 prokaroytes not yet in play –We estimate about 4,800 non-pathogen taxa

The Bergey’s Manual David H. Bergey

Strain Distribution in Collections US Collections / BRCsStrains American Type Culture Collection (ATCC) 4027 USDA ARS Collection (NRRL) 223 European Collections Deutsche Sammlung vor Microoransmen (DSMZ)1302 Culture Collection University Gottenberg (CCUG) 183 Pasteur Institute (CIP) 170 Laboratory for Micrbiology, Gent (LMG) 101 National Collection of Industrial and Marine Bacteria 25 French Collection of Phytopathogens (CFPB) 15 National Collection of Type Cultures (NCTC) 12 National Collection of Phytopathogenic Bacteria 11 Asia Japan Collection of Microorganisms (JCM) 185 Institute of Fermentation, Osaka (IFO) 34 Korean Collection of Type Cultures (KCTC) 28 Institute of Applied Microbiology, Tokyo (IAM) 26 National Institute of Technology And Evaluation (NBRC) 24 All-Russian Collection of Microorganisms (VKM) 13

Estimated Sequencing Rates Target Selection Type Culture Material Sequencing Assembly Rapid Annotation (24 Hours) Metabolic Reconstruction Phenotype Microarrays

Target Selection

Microbial Idol

>2,000 different media Physical Conditions: Temperature (4° - 120°C) pH ( ) Salt (0 - 30%) Light (obligate phototrophs Pressure (few obligate piezophiles) Redox:  Strict anaerobes  Facultative  Microaerobes  Aerobes Culturing by ATCC

Phenotyping by Biolog Carbon Pathways Nitrogen Pathways Sensitivity to Chemicals Osmotic & Ion Effects pH Effects Biosynth. Pathways P S N

Sequencing by JGI FY 06: # Instruments Sanger: : 1 FY 07: # Instruments Sanger: : Gb 45 Gb goal

Automated process consisting of: –Gene calling –Initial annotation of function –Initial metabolic reconstruction Process takes 1-7 hours depending on size and complexity of the genome ~20 genomes per day Rapid Annotation Using Subsystems Technology

Evaluation / Viewing

Feedback Target Selection Sequencing Annotation Metabolic Reconstruction Phenotyping

Status 100 organism pilot - GEBA underway Requesting funding/approval for remainder Target selection about to go live

People JGI Jim Bristow Jonathan Eisen Phil Hugenholtz Nikos Kyrpides Paul Richardson David Bruce MSU Jim Cole George Garrity U GA Barney Whitman UIUC Gary Olsen ATCC David Emmerson Tim Lilburn Biolog Stacy Montgomery John Groat ANL Rick Stevens Folker Meyer Ross Overbeek Veronika Vonstein Hope Matt DeJongh

Technical Feasibility FAQ How many genomes would the project propose to sequence? –About 5000 Who would produce the biomass needed for DNA extraction? –Type culture centers Will the biomass/DNA be available for distribution? –Yes, both the DNA and the libraries could be stored for distribution What throughput is needed for DNA production? –In the beginning of the project ~300 taxa per year to 2000 per yr at the end What throughput is needed for sequencing? –1.2 Gb/yr to 8 Gb/yr finished sequence What combinations of sequencing technologies need to be employed? –Sanger and Pyrosequencing initially What throughput is needed for annotation? –24 hour turnaround from assembled sequence to initial availability Is is possible to have a standard set of phenotype assays given the broad spectrum of organisms and conditions? –We are considering Biolog as a model, but it is too limited How would the genomes be selected and prioritized? –At each cycle we choose genomes (e.g. via 16s) to minimize the diversity gaps Is it necessary to “close” the genomes? –We think no.