jonathan eisen talk at #ievobio 2010

Phylogenomics of microbes: the dark matter of biology

Jonathan A. EisenUC Davis

Talk for iEVOBIOJune 29, 2010

Tuesday, June 29, 2010

Eisen Lab - Phylogenomics of Novelty

Origin of New Functions and

Processes

Species Evolution

Genome Dynamics

•New genes•Changes in old genes•Changes in pathways

•Phylogenetic history•Vertical vs. horizontal descent•Needed to track gain/loss of processes, infer convergence

•Evolvability•Repair and recombination processes•Intragenomic variation


Social Networking in Science


Bacterial evolve


• There are known knowns. These are things we know that we know.

• There are known unknowns. That is to say, there are things that we know we don't know.

• But there are also unknown unknowns. There are things we don't know we don't know.

An homage to Donald Rumsfeld


• Known knowns (background)–rRNA Tree of Life–Genomics–rRNA PCR–Metagenomics

• Known unknowns–GEBA project - past–GEBA project - present–GEBA project - future

• Unknown unknowns?

Outline


Known Knowns 1:

rRNA Tree of Life


rRNA Tree of Life

FIgure from Barton, Eisen et al. “Evolution”, CSHL Press.

Based on tree from Pace NR, 2003.

Archaea

Eukaryotes

Bacteria


The Tree of Life: Three Main Domains

Eukaryotes

Archaea

Bacteria

The Tree of Life

Unrooted Tree of Life from Barton et al. EvolutionTuesday, June 29, 2010

Known Knowns 2:

Genomics and Phylogenomics


Fleischmann et al. 1995


Microbial genomes

From http://genomesonline.orgTuesday, June 29, 2010

Genome Sequences Have Revolutionized Microbiology

• Predictions of metabolic processes

• Better vaccine and drug design

• New insights into mechanisms of evolution

• Genomes serve as template for functional studies

• New enzymes and materials for engineering and synthetic biology


Microbes Run the Planet


4.

Microbes in the world I:rRNA PCR

Perna et al. 2003Tuesday, June 29, 2010

Lateral Transfer

from Doolittle, 1999Tuesday, June 29, 2010

from Lerat et alTuesday, June 29, 2010

Known Knowns 3:

rRNA PCR


Great Plate Count Anomaly

Culturing Microscope

CountCount




CountCount <<<<




CountCount <<<<

DNA


PCR RevolutionExtract DNA

PCR w/ Universal rDNA Primers

Sequence

Align and compareto other rDNAs

Phylogeneticclassification

OTUs Ecology


Uses of rDNA PCRBohannan and Hughes 2003

Hugenholtz 2002


rRNA challenges

• Massive amounts of data from next-gen• Need for full automation but

–Non overlapping–Alignments not always straightforward–BLAST insufficient–Phylogenetic methods that have been automated

still need work• Tree of everything might be useful


Known Knowns 4:

Metagenomics


4.

Microbes in the world I:rRNA PCR

Perna et al. 2003Tuesday, June 29, 2010

Metagenomics

shotgun

clone


Metagenomics Challenge


Metagenomics Challenge

Who is out there?What are they doing?


rRNA phylotyping from metagenomics

Venter et al., 2004


Shotgun Sequencing Allows Use of Alternative Anchors (e.g., RecA)

Venter et al., 2004


0

0.1250

0.2500

0.3750

0.5000

Alphapro

teob

acte

ria

Betap

rote

obac

teria

Gamm

apro

teob

acte

ria

Epsilon

prote

obac

teria

Deltap

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obac

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Cyano

bacte

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Firm

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Actino

bacte

ria

Chloro

biCFB

Chloro

flexi

Spiroch

aete

s

Fuso

bacte

ria

Deinoc

occu

s-Th

erm

us

Eurya

rcha

eota

Crena

rcha

eota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes

Major Phylogenetic Group

EFGEFTuHSP70RecARpoBrRNA

Shotgun Sequencing Allows Use of Other Markers

Venter et al., 2004


Functional Inference from Metagenomics

• Can work well for individual genes• Predicting “community” function is

challenging because treating community as a bag of genes does not work well

• Better to “compartmentalize” data ...


ABCDEFG

TUVWXYZ

Binning challenge


ABCDEFG

TUVWXYZ

Binning challenge

Best binning method: reference genomes


Reference Genomes Coming from Select Environment


ABCDEFG

TUVWXYZ

Binning challenge

No reference genome? What do you do?


ABCDEFG

TUVWXYZ

Binning challenge


Assembly? Composition? Get more references?Tuesday, June 29, 2010

ABCDEFG

TUVWXYZ

Binning challenge


Phylogeny ....Tuesday, June 29, 2010

Metagenomic challenges

• Massive amounts of data from next-gen• Need for full automation but

–Data fragmentary–BLAST insufficient–Automation of phylogenetic methods a bit better for

protein coding genes b/c alignments better–Reference databases incomplete


Known Unknowns 1:

GEBA Past


Microbial genomes

From http://genomesonline.orgTuesday, June 29, 2010

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi

FirmicutesFusobacteria Actinobacteria

Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira

SynergistesDeferribacteres

Thermudesulfobacteria

Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• At least 40 phyla of bacteria

2002

Based on Hugenholtz, 2002


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter


• Genome sequences are mostly from three phyla


2002


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter



• Some other phyla are only sparsely sampled


2002


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter




• Same trend in Archaea


2002


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter




• Same trend in Eukaryotes


2002


The Tree is not Happy



Archaea

Eukaryotes

Bacteria


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter




• Solution I: sequence more phyla

• NSF-funded Tree of Life Project

• A genome from each of eight phyla

Eisen & Ward, PIs


The Tree of Life is Still Angry



Eukaryotes

Bacteria

Archaea


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

• 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

Well sampled phyla


A Genomic Encyclopedia of Bacteria and Archaea (GEBA)


GEBA Pilot Project Overview

• Identify major branches in rRNA tree for which no genomes are available

• Identify branches with a cultured representative in DSMZ

• Grow > 200 of these and prep. DNA• Sequence and finish 100 (covering breadth of

bacterial/archaea diversity)• Annotate, analyze, release data• Assess benefits of tree guided sequencing


GEBA Pilot Project: Components• Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan Eisen,

Eddy Rubin, Jim Bristow)• Project management (David Bruce, Eileen Dalin, Lynne Goodwin)• Culture collection and DNA prep (DSMZ, Hans-Peter Klenk)• Sequencing and closure (Eileen Dalin, Susan Lucas, Alla Lapidus, Mat

Nolan, Alex Copeland, Cliff Han, Feng Chen, Jan-Fang Cheng)• Annotation and data release (Nikos Kyrpides, Victor Markowitz, et al)• Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu, Victor

Kunin, Neil Rawlings, Ian Paulsen, Patrick Chain, Patrik D’Haeseleer, Sean Hooper, Iain Anderson, Amrita Pati, Natalia N. Ivanova, Athanasios Lykidis, Adam Zemla)

• Adopt a microbe education project (Cheryl Kerfeld)• Outreach (David Gilbert)• $$$ (DOE, Eddy Rubin, Jim Bristow)


GEBA and Openness

• All data released as quickly as possible w/ no restrictions to IMG-GEBA; Genbank, etc

• Data also available in Biotorrents (http://biotorrents.net)

• Individual genome reports published in OA “Standards in Genome Sciences (SIGS)”

• 1st GEBA paper in Nature freely available and published using Creative Commons License


http://biotorrents.net

http://biotorrents.net

Known Unknowns 2:

GEBA present


GEBA Lesson 1

rRNA Tree is Useful for Identifying Phylogenetically Novel Genomes


rRNA Tree of Life



Archaea

Eukaryotes

Bacteria


Network of Life

Figure from Barton, Eisen et al. “Evolution”, CSHL Press.


Archaea

Eukaryotes

Bacteria


“Whole Genome” Concatenation Tree w/ AMPHORA

http://bobcat.genomecenter.ucdavis.edu/AMPHORA/See Wu and Eisen, Genome Biology 2008 9: R151


http://bobcat.genomecenter.ucdavis.edu/AMPHORA/





Compare PD in Trees


PD of rRNA, Genome Trees Similar

From Wu et al. 2009 Nature 462, 1056-1060Tuesday, June 29, 2010

http://www.nature.com/nature/journal/v462/n7276/full/nature08656.html


GEBA Lesson 2

Phylogeny-driven genome selection helps discover new genetic diversity


Network of Life



Archaea

Eukaryotes

Bacteria


Protein Family Rarefaction Curves

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


Synapomorphies exist


GEBA Lesson 3

Phylogeny-driven genome selection improves genome annotation


Predicting Function

• Key step in genome projects• More accurate predictions help guide

experimental and computational analyses• Many diverse approaches• Comparative and evolutionary analysis greatly

improves most predictions


Phylogeny vs. Blast

1 2 3 4 5 6

3 5

3

1A 2A 3A 1B 2B 3B

2A 1B

1

1 2

2

2 31

1A 3A

1A 2A 3A

1A 2A 3A

4 6

4 5 6

4 5 6

2B 3B

1B 2B 3B

1B 2B 3B

1A3A

1B 2B3B

12 4

62A

2A

53

5

EXAMPLE BMETHOD

Duplication?

Duplication?

IDENTIFY HOMOLOGS

OVERLAY KNOWNFUNCTIONS ONTO TREE

INFER LIKELY FUNCTIONOF GENE(S) OF INTEREST

ALIGN SEQUENCES

CALCULATE GENE TREE

CHOOSE GENE(S) OF INTEREST

Species 3Species 1 Species 2

ACTUAL EVOLUTION(ASSUMED TO BE UNKNOWN)

EXAMPLE A

Duplication?

Duplication

Ambiguous

Based on Eisen, 1998 Genome Res 8: 163-167.

Many methods focus on “top blast hits”

But much better to build phylogenetic trees of genes and compare to relatives

Allows better integration of evolutionary history (e.g., orthologs and paralogs)


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9521918&query_hl=2




Wu et al. 2005 PLoS Genetics 1: e65. Tuesday, June 29, 2010

http://www.ncbi.nlm.nih.gov/entrez/utils/lofref.fcgi?PrId=4656&uid=16311624&db=pubmed&url=http://genetics.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pgen.0010065

http://www.ncbi.nlm.nih.gov/entrez/utils/lofref.fcgi?PrId=4656&uid=16311624&db=pubmed&url=http://genetics.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pgen.0010065

Most/All Functional Prediction Improves w/ Better Phylogenetic Sampling

• Conversion of hypothetical into conserved hypotheticals

• Improved phylogenomics• Linking distantly related members of

protein families• Improved non-homology prediction


Known Unknowns 3:

GEBA future


GEBA Future 1

How much further should we go?


rRNA Tree of Life



Archaea

Eukaryotes

Bacteria


Phylogenetic Diversity: Sequenced Bacteria & Archaea

From Wu et al. 2009Tuesday, June 29, 2010



Phylogenetic Diversity with GEBA




Phylogenetic Diversity: Isolates




Phylogenetic Diversity: All

From Wu et al. 2009




Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter



species are sparsely sampled• Lineages with no cultured

taxa even more poorly sampled

Well sampled phylaPoorly sampled

No cultured taxaTuesday, June 29, 2010

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter



species are sparsely sampled• Lineages with no cultured taxa

even more poorly sampled

Well sampled phyla

Poorly sampled

No cultured taxaTuesday, June 29, 2010

Uncultured Lineages:Technical Approaches

• Get into culture• Enrichment cultures• If abundant in low diversity ecosystems• Flow sorting• Microbeads• Microfluidic sorting• Single cell amplification


GEBA Future 2

How many gene families are there?


Compare PD in Trees


Gene Families vs PD

0

0.1

0.2

0.3

0.4

0 275 550 825 1100

PD vs. Gene Families (per genome)

PD

/Gen

ome

Gene families / genome


How many protein families?

From Wu et al. 2009

Text

GEBA Genomes PD/Genome ~0.1

PFAMs/Genome ~1000

PFAMs/PD~10000

Total PFAMS~10,000,000




Caveats (of many)

• Novel protein families per genome likely taxon specific

• Parameters other than PD clearly important

• Does not include viruses, eukaryotes


GEBA Future 3

Need to better leverage improved phylogenetic sampling


Example 1: Protein Family Space

• Much less biased sampling of protein family space now available

• Need to rebuild / reassess many protein family databases (e.g., HMMs)

• Structural space


Example 2: Experiments


Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter


As of 2002



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter


• Experimental studies are mostly from three phyla

As of 2002



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter


• Experimental studies are mostly from three phyla

• Some studies in other phyla

As of 2002



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter




• Same trend in Eukaryotes

As of 2002



Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter




• Same trend in Viruses

As of 2002



0.1

Acidobacteria

Bacteroides

Fibrobacteres

Gemmimonas

Verrucomicrobia

Planctomycetes

Chloroflexi

Proteobacteria

Chlorobi


Cyanobacteria

Chlamydia

Spriochaetes

Deinococcus-Thermus

Aquificae

Thermotogae

TM6OS-K

Termite GroupOP8

Marine GroupAWS3

OP9

NKB19

OP3

OP10

TM7

OP1OP11

Nitrospira



Chrysiogenetes

Thermomicrobia

Dictyoglomus

Coprothmermobacter

Tree based on Hugenholtz (2002) with some modifications.

Need experimental studies from across the tree too


Example 3: Improving the tree

• To make best use of GEBA data we need a better tree


Wh

Concatenated alignment “whole genome tree” built using AMPHORA


Wh

Whole genome tree built using AMPHORAby Martin Wu and Dongying Wu

Why does the

tree matter?


Many Alternatives to Concatenation

• Gene presence/absence• Supertrees / consensus methods• Separate phylogeny of genes and then

integration of results (e.g., networks)• Models that incorporate gain/loss as well as

gene phylogeny


Example 4: Metagenomic Analysis


0

0.1250

0.2500

0.3750

0.5000

Alphapro

teob

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Betap

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teria

Gamm

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Epsilon

prote

obac

teria

Deltap

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Cyano

bacte

ria

Firm

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s

Actino

bacte

ria

Chloro

biCFB

Chloro

flexi

Spiroch

aete

s

Fuso

bacte

ria

Deinoc

occu

s-Th

erm

us

Eurya

rcha

eota

Crena

rcha

eota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes



Phylogeny for Typing and Binning

Venter et al., 2004


0

0.1250

0.2500

0.3750

0.5000

Alphapro

teob

acte

ria

Betap

rote

obac

teria

Gamm

apro

teob

acte

ria

Epsilon

prote

obac

teria

Deltap

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obac

teria

Cyano

bacte

ria

Firm

icute

s

Actino

bacte

ria

Chloro

biCFB

Chloro

flexi

Spiroch

aete

s

Fuso

bacte

ria

Deinoc

occu

s-Th

erm

us

Eurya

rcha

eota

Crena

rcha

eota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes



Venter et al., 2004

Should improve with better genomic sampling



0

0.1250

0.2500

0.3750

0.5000

Alphapro

teob

acte

ria

Betap

rote

obac

teria

Gamm

apro

teob

acte

ria

Epsilon

prote

obac

teria

Deltap

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obac

teria

Cyano

bacte

ria

Firm

icute

s

Actino

bacte

ria

Chloro

biCFB

Chloro

flexi

Spiroch

aete

s

Fuso

bacte

ria

Deinoc

occu

s-Th

erm

us

Eurya

rcha

eota

Crena

rcha

eota

Sargasso Phylotypes

Wei

ght

ed %

of

Clo

nes



Venter et al., 2004

Only improved a little



How to improve phylogenetic analysis of metagenomic data

• Better phylogenetic and OTU methods for fragmented data

• Better assessment of which genes to use?

• More automation of all methods


Phylogenetic challenge

How place all in one tree?How identify OTUs including all fragments?Can you analyze more than 1 gene family at a time?


Approach 1:Place Reads on Reference Tree

• Examples–AMPHORA (Wu and Eisen)–PPlacer (Erik Matsen)–FastTree (Morgan Price)

• General approach–Precompute reference tree for full length sequences–Place individual reads on reference tree–Merge trees


Variants

• Use concatenated alignment of markers not just individual genes (Steven Kembel)

• Apply to OTU identification not just classification (Thomas Sharpton)

• CoBinning: look for linkage among fragments/genes (Aaron Darling)





• More automation


New “Marker Genes”

• 100 representative genomes, including many GEBAs

• MCL gene families• Identify gene families w/

–High universality–High uniformity of copy number–Phylogenetic tree similar to “whole genome tree”


0 1 2 3 4 5 6

rRNA16SruvBnusArplBpurArpsJsecYrpsIpyrHrpsErplPrplNrpsCruvArplFrplAserSrplKrpsKpriAsmpBrpsGguaArpsQrpsLrplUrplOrpsMinfCrplSrplVrplCrpsPrplErplTrplLrplQrpsHmraWrpsOrpsBrplIrplMrplRttffrrtsfrplDradArpsStrmDcoaErpmA

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

nusArpsCrpsEpriArplBsecY

rRNA16SrpsJrpsBruvBguaArplNserSrplFfrrrplArplErplCinfCrplDrplKpurAradAruvArpsMpyrHrplIrplMrpsGrpsLmraWrpsIttfrplStrmDtsfrplUrpsKrpsPrplOrplTrplVrpsSrplPrpsOsmpBrpsHrplQrplRrpsQrplLrpmAcoaE

Ribosomal protein Transcription/translation related protein DNA repair protein Protein of other functionAMPHORA marker

Distance between the genome tree and 100 random trees (average ± standard deviation)

NODAL distance SPLIT distance

Distances between gene trees and the AMPHORA concatenated genome tree


Screen gene markers for different taxonomic groups

phylum Genome Number

Gene NumberActinobacteria 63 267783Alphaproteobacteria

94 347287Betaproteobacteria

56 266362Gammaproteobacteria

126 483632Deltaproteobacteria

25 102115Epislonproteobacteria

18 33416Bacteriodes 25 71531Chlamydae 13 13823Chloroflexi 10 33577Cyanobacteria 36 124080Firmicutes 106 312309Spirochaetes 18 38832Thermi 5 14160Thermotogae 9 17037


Keep only the families with:

Universality * Evenness * monophyly >= 90*90*90

Phylogenetic group Genome Number Gene Number Maker Candidates

Archaea 62 145415 102

Actinobacteria 63 267783 136

Alphaproteobacteria 94 347287 142

Betaproteobacteria 56 266362 294

Gammaproteobacteria 126 483632 141

Deltaproteobacteria 25 102115 44

Epislonproteobacteria 18 33416 446

Bacteriodes 25 71531 179

Chlamydae 13 13823 561

Chloroflexi 10 33577 140

Cyanobacteria 36 124080 532

Firmicutes 106 312309 80

Spirochaetes 18 38832 72

Thermi 5 14160 727

Thermotogae 9 17037 646





• More automation


AMPHORA

Guide treeTuesday, June 29, 2010

Phylogenetic Binning Using AMPHORA

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0.1

0.2

0.3

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Alph

apro

teob

acteria

Betapr

oteo

bacter

ia

Gammap

roteob

acteria

Deltapr

oteo

bacter

ia

Epsil

onpr

oteo

bacter

ia

Uncla

ssified

Pro

teob

acteria

Cyan

obac

teria

Chlamyd

iae

Acidob

acteria

Bacter

oide

tes

Actin

obac

teria

Aquific

ae

Plan

ctom

ycetes

Spiro

chae

tes

Firmicu

tes

Chloro

flexi

Chloro

bi

Uncla

ssified

Bac

teria

dnaGfrrinfCnusApgkpyrGrplArplBrplCrplDrplErplFrplKrplLrplMrplNrplPrplSrplTrpmArpoBrpsBrpsCrpsErpsIrpsJrpsKrpsMrpsSsmpBtsf

AMPHORA - each read on its own treeTuesday, June 29, 2010

Zorro

• http://sourceforge.net/projects/probmask/• ZORRO is a probabilistic masking program

that assigns confidence scores to each column in a multiple sequence alignment. These scores can then be used to account for alignment accuracy in phylogenetic inference pipelines

• Wu, Chatterji, Eisen submitted


http://sourceforge.net/projects/probmask/

http://sourceforge.net/projects/probmask/

Conclusions

• Phylogeny-driven sampling produces many benefits immediately

• For the most benefits to come, we need to re-direct many informatics efforts to take advantage of less biased data

• Still a long way away from full benefits• Note - most of the benefits can come from

(aack) - unfinished genomes


MICROBES


A Happy Tree of Life


jonathan eisen talk at #ievobio 2010

Education