GEBA A genomic encyclopedia of bacteria and archaea

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GEBAA genomic encyclopedia of bacteria and
archaea

• Jonathan A. Eisen
• JGI User Meeting 2009

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Nothing in biology makes sense except in the
light of evolution. T. Dobzhansky (1973)
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rRNA Tree of Life
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The Tree is not Happy
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From http//genomesonline.org
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As of 2002

• At least 40 phyla of bacteria

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Based on Hugenholtz, 2002
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As of 2002

• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Based on Hugenholtz, 2002
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As of 2002

• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Based on Hugenholtz, 2002
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As of 2002

• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
• Same trend in Archaea

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Based on Hugenholtz, 2002
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Need for Tree Guidance Well Established

• Common approach within some eukaryotic groups
• Many small projects funded to fill in some
  bacterial or archaeal gaps
• Phylogenetic gaps in bacterial and archaeal
  projects commonly lamented in literature

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• NSF-funded Tree of Life Project
• A genome from each of eight phyla

• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
• Solution I sequence more phyla

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Eisen, Ward, Badger, Wu, Wu, et al.
Chloroflexi
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Bacterial aTOL Project AIMS

• Improve resolution of deep branches in the
  bacterial tree
• Launch biological studies of these phyla and
  discover functional novelty
• Leverage data for interpreting environmental
  surveys

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T. roseum genome
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The Tree of Life is Still Angry
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Within Phyla Diversity Immense

• Each phyla represents billions of years of
  evolution
• Some have hundreds of major lineages
• New lineages are being discovered all the time
• Most branches within most phyla have few or no
  genomes

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Major Lineages of Actinobacteria
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Additional Impetus for Tree Guided Projects

• Suggestion to sequence all bacteria and archaea
  in Bergeys Manual (Stevens et al)
• Success in sequencing genomes from across the
  tree in animals
• Multiple government reports suggest a more
  systematic approach to sequencing is needed

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• At least 100 phyla of bacteria
• Genome sequences are mostly from three phyla
• Most phyla with cultured species are sparsely
  sampled
• Lineages with no cultured taxa even more poorly
  sampled
• Solution - use tree to really fill gaps

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Well sampled phyla
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http//www.jgi.doe.gov/programs/GEBA/pilot.html
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GEBA Pilot Project Overview

• Select 200 organisms using tree
• Develop high throughput pipeline for strain
  growth and DNA preparation
• Sequence and finish 100
• Annotate, analyze, release data
• Assess benefits of tree guided sequencing

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GEBA Pilot I Selecting Targets
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GEBA Pilot II The Importance of Project
Management
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• GEBA Project Flowchart

Annotation
Project Initiation
Sequencing
IMG1
Draft Sequencing and Assembly1
GEBA Proposal
Complete Genome GenBank Submission1
Shotgun Genome GenBank Submission1
Scientific and Technical Review1
OK?
OK?
IMG ER1
IMG ER1
Finish Sequencing and Assembly2
Negotiate Scope of Work
Gene-QA1
Draft Annotation3
Receive Starting Material1
Finish Annotation3
OK?
1 PGF 2 LANL 3 ORNL
David Bruce, Lynne Goodwin et al
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GEBA Pilot III Partnership with DSMZ
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GEBA Biggest ChallengeGetting DNA

• Getting quality DNA is biggest bottleneck
• Solution Beg Borrow and Steal
• DSMZ offered to do for free
• ATCC is doing a small number for a fee
• In discussions with other PCC and other
  collections

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Quantification gel of the genomic DNA isolated
from Conexibacter woesei (DSM 14684T)
Microorganisms
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Lane 1 c(?-Marker) 15 ng Lane 2 c(?-Marker)
30 ng Lane 3 c(?-Marker) 50 ng Lane 4 DNA
Molecular Weight Marker II (Roche 236250) Lane 5
DSM 13279, Collinsella stercoris Lane 6 DSM
43043, Intrasporangium calvum Lane 7 DSM 18053,
Dyadobacter fermentans Lane 8 DSM 20476, Slackia
heliotrinireducens
Lane 9 DSM 18081, Patulibacter
minatonensis Lane 10 DSM 14684, Conexibacter
woesei Lane 11 DSM 11002, Dethiosulfovibrio
peptidovorans Lane 12 DSM 11551, Halogeometricum
borinquense Lane 13 DNA Molecular Weight Marker
II (Roche 236250) Lane 14 c(?-Marker) 125
ng Lane 15 c(?-Marker) 250 ng Lane 16
c(?-Marker) 500 ng
Conexibacter woesei (DSM 14684T) was taken from
the German Collection of Microorganisms and Cell
Cultures (DSMZ). The genomic DNA was isolated
using the Qiagen Genomic 500 DNA Kit (Qiagen
10262). The genomic DNA was 10-250 kb in size as
determined by Pulsed Field Gel Electrophoresis
(PFGE). The bulk of DNA had a size of 50-250 kb
(see attached PFGE image). The DNA concentration
is 500 ng/µl as estimated from the gel.
Spectrophotometric measurements yielded a DNA
concentration of 450 µg/ml 300 µl of genomic DNA
are shipped (150 µg).
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GEBA Pilot IV Sequencing, Annotation, Data
Release
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Current Status

• gt100 in progress
• GEBA 56 (focus of first paper)
• 34 finished genomes
• 55 submitted to Genbank
• Released to IMG-GEBA page and JGI-FTP site
• All data is completely Open for anyone to use

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IMG/GEBA
http//img.jgi.doe.gov/cgi-bin/geba/main.cgi
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Adopt a Microbe
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GEBA Pilot IV Assess Benefits of GEBA56

• All genomes have some value
• But what, if any, is the benefit of tree-guided
  sequencing over other selection methods

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Why Increase Taxonomic Coverage II?

• Gene discovery
• Annotation, functional prediction
• Metagenomic analysis
• Mechanisms of diversification
• Species phylogeny and classification

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Value of diverse genomes I Gene discovery

• Premise
• New genomes frequently contain genetic novelty
• Phylogenetic diversity of a genome should be
  correlated to novelty
• Caveat
• Does lateral gene transfer wipe out contribution
  of phylogenetic diversity to novelty?

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Protein Family Rarefaction Curves

• Take data set of multiple complete genomes
• Identify all protein families using MCL
• Plot of genomes vs. of protein families

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Number of proteins
Total Gene Number
Genome Number
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Novelty 2 - Structural Novelty

• Of the 17000 protein families in the GEBA56, 1800
  are novel in sequence (Wu)
• Structural modeling suggests many are
  structurally novel too (D'haeseleer)
• 372 being crystallized by the PSI (Kerfeld)

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Novelty 3

• Diversity within known families

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Transporter Profiles
Sebaldella termitidis ATCC 33386 has 2x number of
sugar PTS transporters of any genome
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Novelty 4

• Unusual distribution patterns

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Shotgun Sequencing Detects More Diversity than
PCR-methods
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First Bacterial Actin Related Protein
First found by V. Kunin, Structure Analysis by
Patrik D. et al
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Most Closely Related to ARP8
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Value of 100 diverse genomes II Annotation

• Premise
• Increased phylogenetic coverage should improve
  our ability to annotate genes in other (e.g.,
  reference/model genomes)

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Annotation Improves

• Conversion of hypothetical into conserved
  hypotheticals
• Linking distantly related members of protein
  families
• Non-homology functional prediction methods

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Linking Protein Families Improved
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Fusion Based Predictions Improved
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Improving Rosetta Stone Predictions
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Value of 100 diverse genomes III Metagenomics

• Premise
• Increased sampling of diverse genomes should
  improve many aspects of metagenomic analysis
• To test
• Annotation
• Binning

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Metagenomic Annotation Improves (Slightly)
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Compositional Binning Improves (Slightly)
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Phylogenetic Binning Improves Slightly
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Value of 100 diverse genomes V Phylogeny
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16s Says Hyphomonas is in Rhodobacteriales
Badger et al. 2005
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WGT Says Its Related to Caulobacterales
Badger et al. 2005
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GEBA - After the Pilot
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PD of sequenced organisms
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PD with GEBA
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• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Most phyla with cultured species are sparsely
  sampled
• Lineages with no cultured taxa even more poorly
  sampled

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Well sampled phyla
Poorly sampled
No cultured taxa
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As of 2002

• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
• Same trend in Viruses

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Based on Hugenholtz, 2002
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As of 2002

• At least 40 phyla of bacteria
• Genome sequences are mostly from three phyla
• Some other phyla are only sparsely sampled
• Same trend in Microbial Eukaryotes

Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
Chloroflexi
Based on Hugenholtz, 2002
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Need experimental studies from across the tree too
Acidobacteria
Bacteroides
Fibrobacteres
Gemmimonas
Verrucomicrobia
Planctomycetes
0.1
Chloroflexi
Tree based on
Hugenholtz (2002)
with some
modifications.
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MICROBES
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A Happy Tree of Life