Production Genomics Facility DOE JGI Production Genomics Facility Opened in 1999 ~240 UC Employees ~$66M Annual Budget ~47 Tb for 2012 >37 Human genomes / day Mission: “User facility providing high-throughput DNA sequencing & analysis in support of DOE missions in alternative energy, carbon cycling & bioremediation.”

{ { { Evolution of JGI Sequencing Platforms Output (Tb) Staff (#) FY2009 49 Units 24 FTEs $11M 1Tb FY2010 22 Units 15 FTEs $11M 6Tb FY2011 15 Units 9 FTEs $8M 29Tb { { { Staff (#) Output (Tb) 3730 Budget ($M) GAii 454 GAii Hiseq 454 GAii Hiseq 454

JGI Science Programs

Community Sequencing Program DOE JGI Community Sequencing Program >700 Users

SCIENCE GOALS CSP 1 CSP 2 CSP 3 CSP 4 FOCUS AREA CSP 1 CSP 2 CSP 3

Program Science 2012 - Focus Areas PLANT-MICROBE INTERACTIONS Dangl Arabidopsis Boechera Miscanthus Tuscan Poplar Agave Gross Andersson Lichen CARBON CYCLING SOIL Fierer Grassland soil carbon cycling Brodie Mediterranean grassland Mohn American forest soils Rodrigues Amazon deforestation AQUATIC CARBON CYCLING McMahon Crystal Bog Lake McKay Lake Erie Gilbert Gulf of Mexico Moran Ocean heterotrophs Dangl: profiling of arabidopsis genotypes and hybrids for gene mapping; profiling of Boechera stricta and Miscanthus as well as prairie site subjected to climate change manipulations. Poplar: profiling of genotyped trees in Oregon; metagenome sequencing of 24 samples (4 genotypes X 2 locations X 3 replicates) Lichen: sequencing of all three primary players as well as metagenome sequencing to characterize others; metatranscriptome sequencing to characterize eukaryotic stress responses. Agave: rhizosphere and phyllosphere of cultivated and wild species, including single cell characterization of endophytes Fierer: Shallow sequencing of 60 soil samples from different treatments (Co-Is Gilbert, Knight, Earth Microbiome Project) Brodie: Deep sequencing of 2 soils (different seasons) and 30 isolates McMahon: 100 community DNA samples for comparative metagenomics (Co-Is Malmstrom, Gilbert, Earth Microbiome Project) McKay: 4 metagenomes + 4 metatranscriptomes Moran: Mostly mutant screening but also some metatranscriptomics of environmental samples Gilbert: 100 samples from oil spill time series (Earth Microbiome Project)

Microbial Genomics Nikos Kyrpides Microbial Genomics and Metagenomics Programs DOE Joint Genome institute 7

Historical perspective Do we need more sequencing? Overview of the talk Historical perspective Do we need more sequencing? Major Transitions in Genomics

The Quest for Darwin’s Grail [unlike understanding an electron] "To understand life [unlike understanding an electron] you must understand its history" Carl R. Woese

Darwin's Dream "The time will come I believe, though I shall not live to see it, when we shall have fairly true genealogical trees of each great kingdom of nature" [in a letter to T.H. Huxley] Charles Darwin, 1857

Quotes from “The Microbial World” “It is a waste of time to attempt a natural system of classification for bacteria” 1st Edition –1957 “The only possible conclusion is, accordingly that the ultimate scientific goal of biological classification cannot be achieved in the case of bacteria” 2nd Edition –1963 “For bacteria, the general course of evolution will probably never be known, and there is simply not enough objective evidence to base their classification on phylogenetic grounds” 3rd Edition – 1970 4th Edition – 1977 Verbatim repeat of the 3rd edition

1978 – 1st Universal Tree of Life

History of Genomics

Carl Woese 1998 Microbial Genomics “Genome sequencing has come of age, and genomics will become central to microbiology’s future. It may appear at the moment that the human genome is the main focus and primary goal of genome sequencing, but do not be deceived. The real justification in the long run, is microbial genomics.” Carl Woese 1998

UNDERSTANDING vs INFORMATION GREAT CHALLENGES P. Chain et al. Science, 2009 UNDERSTANDING vs INFORMATION 1995-2009 2010-2015 Finished 1000 3000 Draft 10000 Genes 8 Million 52 Million 2010-2013 3,000 20,000 92 Million 16

Why we need more sequencing Ribokinase family Fructokinase family 2-dehydro-3-deoxy glucokinase family

We need to change this … Genome projects 2000 Genome projects 2010 11% 71 bacterial genomes Genome projects 2000 5500 bacterial genomes Genome projects 2010 11%

Many Phyla – many gaps Poor sequence coverage mainly due to lack of isolates, but many gaps have unsequenced representatives Norman Pace

99% of microorganisms are not culturable with present methods. What about the Uncultured majority? Culturable Unculturable 99% of microorganisms are not culturable with present methods.

The road to success in Metagenomics is through Microbial Genomics Metagenomic Analysis Acid Mine Drainage Sargasso Sea Termite Hindgut Human Gut Soil Reference Genomes Species complexity 1 10 100 1000 1000s 10000 Binning ? The road to success in Metagenomics is through Microbial Genomics Source: Susannah Tringe, JGI 21

Availability of Reference Genomes Acid Mine Drainage Human gut Marine Termite Gut Soil Reference Genomes 100% 60% 50% 40% 20% 1% ? 22

Genomes and Metagenomes Microbial environments Genome reconstruction Single cells Cultured microbes Number of Organisms Coverage of Cultured Microbes with Genome Projects 16S rRNA Distance Known diversity of cultured vs uncultured organisms Number of Organisms 16S rRNA Distance

GEBA Goal: Filling in the gaps in sequencing along the bacterial and archaeal branches of the Tree of Life Status: >120 Complete >100 Draft >100 In progress Wu et al. Nature 2009 http://img.jgi.doe.gov/GEBA

Improved Gene annotation and characterization of hypothetical gene families based on novel gene fusions: 7.7 times more fusion events than any other randomly selected 56 genomes novel gene neighbourhoods: 4.3 times more novel gene neighbourhoods than any other randomly selected 56 genomes novel connections of protein families: 47 times more novel connections of protein families than any other randomly selected 56 genomes Mavromatis Amrita Pati Wu et al. Nature 2009

Rob Knight 25K OTUs from cultured (70%), 10K from uncultured (30%). If the density of OTUs is the same between cultuted and unclutured We would expect around 140K OTUs from the uncultured 15% 25K 85% ~ 35,000 OTUs Wu et al. Nature 2009

The Microbial Earth Project STEERING COMMITTEE Nikos Kyrpides, DOE-JGI George Garrity, Names4Life Hans-Peter Klenk, DSMZ Phil Hugenholtz, JGI The Microbial Earth Project Victor Kunin, JGI Dino Liolios, GOLD

Microbial Earth Project

Pangenomes 10 Prochlorococcus marinus Pangenome 17 Listeria monocytogenes Pangenome 17 15 Staphylococcus aureus Pangenome

Strain / species diversity

14765 2733 = 5.4 10434 5820 = 1.8

Metagenome Analysis with Pangenomes Reference Genome Best Blast Hit Pangenome Synteny blocks

HOW REPRESENTATIVE OF THE SPECIES IS THE PANGENOME? PANGENOMES HOW REPRESENTATIVE OF THE SPECIES IS THE PANGENOME? 1. How representative is the pangenome phylogenetically? PANGENOME ☺ 16S DIVERSITY 2. How representative is it geographically? 3. How representative is the habitat coverage? ☺

PARADIGM SHIFT 1960-1990 1990-2010 16S RNA 2010-2020 Genomes Pangenomes 35

Microbial Genomics Program Cell isolation Amplification 16S Screening Sequencing