UC Davis EVE161 Lecture 16 by @phylogenomics

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014
Lecture 16:
EVE 161: 
Microbial Phylogenomics
!
Lecture #16:
Era IV: Shotgun Metagenomics
!
UC Davis, Winter 2014
Instructor: Jonathan Eisen
!1

Where we are going and where we have been
!Previous lecture:
! 15: Era IV: Shotgun Metagenomics
! Current Lecture:
! 16: Era IV: Metagenomic Diversity and
Binning
! Next Lecture:
! 17: Era IV: Metagenomic Case Study
!2

Era IV: Genomes in the environment
Era IV:
Diversity from Metagenomics

A Very Simple System

Pierce’s Disease
!5

• Obligate xylem feeder
• Transmits Xylella between
plants
• Much like mosquitoes
transmit malarial pathogen
• Only animal listed as
possible “bioterror” agent
by US DHS
!6
Glassy winged sharpshooter
GLASSY-WINGED
SHARPSHOOTERASeriousThreattoCaliforniaAgriculture
FROM THE
UNIVERSITY OF CALIFORNIA’S
PIERCE’S DISEASE RESEARCH AND
EMERGENCY RESPONSE TASK FORCE
This informational brochure was produced by ANR
Communication Services for the University of Califor-
nia Pierce’s Disease Research and Emergency
Response Task Force. You may download a copy of the
brochure from the Division of Agriculture and Natural
Resources web site at http://danr.ucop.edu or from the
Communication Services web site at
http://danrcs.ucdavis.edu.
For local information, contact your UC Cooperative
Extension farm advisor:
Adults
Egg masses
Glassy-winged Sharpshooter
Generalized Lifecycle
100
80
60
40
20
0
Jan.
Mar.
May
July
Sept.
Nov.
Glassy-winged sharpshooters overwinter as adults
and begin laying egg masses in late February
through May. This first generation matures as
adults in late May through late August. Second-
generation egg masses are laid starting in mid-
June through late September, which develop into
over-wintering adults.

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !7
Xylem feeding insects also very successful

Xylem and Phloem
From
Lodish
et al.
2000
!8

Animal nutrition
• Xylem is frequently missing essential amino acids,
vitamins and Co-Factors, and has only small
amounts of carbon skeletons
!9

Plant response to sap feeders
• Possible solutions to no aa, vitamins, etc in xylem
! Eat other things
! Evolve metabolic pathways to synthesize missing nutrients
! Find some poor sap to make the stuff for you
!10

Moran N. A. PNAS 2007;104:8627-8633
©2007 by National Academy of Sciences
!11
Sharpshooter:
Cuerna sayi
bacteriomes
symbionts in their bacteriomes
Moran et al. 2003 Environ. Microbiol.
Moran et al. 2005 Appl. Environ. Microbio
Candidatus “Baumannia cicadellinicola” (Gammaproteobacteria)
Candidatus “Sulcia muelleri” (Bacteroidetes)
D Takiya
10!m
“Candidatus Baumannia cicadellinicola” (Gammaproteoba
in “red” portion of bacteriome of Homalodisca vitripennis
N=host nucleus B=Bacteriocyte membrane E=Endosym
Irregu
~2 !m

DNA
extraction
PCR
Sequence
rRNA genes
Sequence alignment = Data matrixPhylogenetic tree
PCR
rRNA1
Yeast
Makes lots of
copies of the
rRNA genes
in sample
E. coli
Humans
A
T
T
A
G
A
A
C
A
T
C
A
C
A
A
C
A
G
G
A
G
T
T
C
rRNA1
E. coli Humans
Yeast
!12
rRNA1
5’ ...TACAGTATAGGTG
GAGCTAGCGATCGAT
CGA... 3’
PCR and phylogenetic analysis of rRNA genes
bacteriomes
r two obligate
bacteriomes
Moran et al. 2005 Appl. Environ. Microbiol.
cola” (Gammaproteobacteria)
roidetes)
D Takiya
mm
domen of H. vitripennis

Baumania is close relative of Buchnera symbionts of aphids
Sharpshoote
Aphids
Aphids
Aphids
Ants
Flies
!13
Wu et al. 2006 PLoS Biology 4: e188.

Baumania is close relative of Buchnera symbionts of aphids
Sharpshoote
Aphids
Aphids
Aphids
Ants
Flies
!14

Sharpshooter Shotgun Sequencing
shotgun
Collaboration with Nancy Moran’s
lab

Higher Evolutionary Rates in
Endosymbionts
Wu et al. 2006 PLoS Biology 4: e188. Collaboration with Nancy Moran’ s Lab

Variation in Evolution Rates 
Correlated with Repair Gene Presence
MutS MutL
+ +
+ +
+ +
+ +
_ _
_ _
Wu et al. 2006 PLoS Biology 4: e188. Collaboration with Nancy Moran’ s Lab

Polymorphisms in Metapopulation
• Data from ~200 hosts

–104 SNPs

–2 indels

• PCR surveys show that this
is between host variation

• Much lower ratio of
transitions:transversions
than in Blochmannia

• Consistent with absence of
MMR from Blochmannia

Predict metabolic networks from Genome
Wu et al.
2006 PLoS
Biology 4:
e188.
!24

Baumannia is a Vitamin and Cofactor Producing Machine
Wu et al.
2006 PLoS
Biology 4:
e188.
!25
VITAMIN AND
COFACTOR
PRODUCING MACHINE

Baumannia is a Vitamin and Cofactor Producing Machine
Wu et al.
2006 PLoS
Biology 4:
e188.
!26
NO PATHWAYS FOR
ESSENTIAL AMINO  
ACID SYNTHESIS

???????

Commonly Used Binning Methods 
Did not Work Well
• Assembly

–Only Baumannia generated good contigs

• Depth of coverage

–Everything else 0-1X coverage

• Nucleotide composition

–No detectible peaks in any vector we looked at

A
B
C
D
E
F
G
T
U
V
W
X
Y
Z
Binning challenge
!30

A
B
C
D
E
F
G
T
U
V
W
X
Y
Z
Binning challenge
Best binning method: reference genomes
!31

A

B

C

D

E

F

G
T

U

V

W

X

Y

Z
Binning challenge
No reference genome? What do you do?

!
Phylogeny ....

DNA
extraction
PCR
Sequence
rRNA genes
Sequence alignment = Data matrixPhylogenetic tree
PCR
rRNA1
rRNA2
Makes lots of
copies of the
rRNA genes
in sample
rRNA1
5’ ...ACACACATAGGTG
GAGCTAGCGATCGAT
CGA... 3’
E. coli
Humans
A
T
T
A
G
A
A
C
A
T
C
A
C
A
A
C
A
G
G
A
G
T
T
C
rRNA1
E. coli Humans
rRNA2
!33
rRNA2
5’ ...TACAGTATAGGTG
GAGCTAGCGATCGAT
CGA... 3’
PCR and phylogenetic analysis of rRNA genes
3)/,0+
bacteriomes
r two obligate
bacteriomes
Moran et al. 2005 Appl. Environ. Microbiol.
cola” (Gammaproteobacteria)
roidetes)
D Takiya
mm
domen of H. vitripennis

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014Wu et al. 2006 PLoS Biology 4: e188.

CFB Phyla

Sulcia makes essential amino acids
!38

Sulcia makes essential amino acids
!39
ESSENTIAL AMINO  
ACID PRODUCING
MACHINE

Baumannia makes vitamins and cofactors
Sulcia makes amino acids

Phylogeny & Metagenomes

• Why analyze protein coding “marker” genes in
metagenomic data?
•

AMPHORA: AutoMated PHylogenOmic infeRence
Wu and Eisen, 2008. Genome Biology 2008, 9:R151 !
doi:10.1186/gb-2008-9-10-r151 http://genomebiology.com/2008/9/10/R151

Choosing Marker Genes
• How choose the marker genes to use?
!

Choosing Marker Genes
• How choose the marker genes to use?
!
• “We selected these proteins because they are universally
distributed in bacteria; the vast majority of them exist as
single copy genes within each genome; and they are
housekeeping genes that are involved in information
processing (replication, transcription, and translation) or
central metabolism, and thus are thought to be relatively
recalcitrant to lateral gene transfer [19].”

Identification and alignment of metagenomic reads?
• How to identify and rapidly align reads that correspond to
each of the markers selected?

Identification and alignment of metagenomic reads?
• How to identify and rapidly align reads that correspond to
each of the markers selected?
!
• Prealign representatives from genomes
• Manually curate to build a “mask”
• Create “Hidden markov model” of the alignment
• Use HMM to search for, and align from metagenomes
!
• For more on HMMs see “What is a Hidden Markov Model” by Sean Eddy. http://www.nature.com/nbt/journal/v22/
n10/full/nbt1004-1315.html

HMMs and Profiles
• Useful guide here http://compbio.soe.ucsc.edu/
ismb99.handouts/KK185FP.html
Proﬁle HMM

0.2
Kineococcus radiotolerans SRS30216
Nitrosomonas eutropha C91
Flavobacterium psychrophilum JIP02 86
Saccharopolyspora erythraea NRRL 2338
Magnetospirillum magneticum AMB 1
Bacillus subtilis subsp subtilis str 168
Acidobacteria bacterium Ellin345
Rhodopirellula baltica SH 1
Mycobacterium bovis BCG str Pasteur 1173P2
Escherichia coli CFT073
Bacillus thuringiensis serovar konkukian str 97 27
Synechococcus sp CC9902
Leptospira interrogans serovar Lai str 56601
Rickettsia conorii str Malish 7
Helicobacter pylori J99
Shigella flexneri 5 str 8401
Bordetella pertussis Tohama I
Mycobacterium sp JLS
Colwellia psychrerythraea 34H
Lawsonia intracellularis PHE MN1 00
Prochlorococcus marinus str MIT 9303
Streptomyces coelicolor A3 2
Lactobacillus helveticus DPC 4571
Yersinia pestis Nepal516
Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis
Haemophilus influenzae 86 028NP
Campylobacter jejuni subsp jejuni 81116
Burkholderia pseudomallei K96243
Borrelia burgdorferi B31
Serratia proteamaculans 568
Tropheryma whipplei TW08 27
Clostridium perfringens SM101
Anaplasma phagocytophilum HZ
Psychrobacter arcticus 273 4
Fusobacterium nucleatum subsp nucleatum ATCC 25586
Rickettsia bellii OSU 85 389
Dehalococcoides ethenogenes 195
Buchnera aphidicola str Cc Cinara cedri
Candidatus Ruthia magnifica str Cm Calyptogena magnifica
Chlamydophila caviae GPIC
Roseobacter denitrificans OCh 114
Shewanella denitrificans OS217
Buchnera aphidicola str APS Acyrthosiphon pisum
Geobacillus thermodenitrificans NG80 2
Parabacteroides distasonis ATCC 8503
Corynebacterium jeikeium K411
Yersinia pseudotuberculosis IP 32953
Francisella tularensis subsp tularensis SCHU S4
Streptococcus pyogenes MGAS315
Zymomonas mobilis subsp mobilis ZM4
Bacillus anthracis str Ames
Shewanella frigidimarina NCIMB 400
Streptococcus gordonii str Challis substr CH1
Thiomicrospira crunogena XCL 2
Streptococcus pyogenes str Manfredo
Pseudomonas putida KT2440
Mycobacterium leprae TN
Staphylococcus saprophyticus subsp saprophyticus ATCC 15305
Shewanella sp MR 7
Rhodopseudomonas palustris HaA2
Geobacter sulfurreducens PCA
Lactococcus lactis subsp cremoris SK11
Nitrosococcus oceani ATCC 19707
Rhodopseudomonas palustris BisB18
Synechococcus elongatus PCC 6301
Pelotomaculum thermopropionicum SI
Synechocystis sp PCC 6803
Sorangium cellulosum So ce 56
Wolinella succinogenes DSM 1740
Moorella thermoacetica ATCC 39073
Gluconobacter oxydans 621H
Mycoplasma capricolum subsp capricolum ATCC 27343
Actinobacillus succinogenes 130Z
Baumannia cicadellinicola str Hc Homalodisca coagulata
Pseudomonas syringae pv syringae B728a
Hahella chejuensis KCTC 2396
Gramella forsetii KT0803
Brucella abortus biovar 1 str 9 941
Ochrobactrum anthropi ATCC 49188
Campylobacter fetus subsp fetus 82 40
Burkholderia mallei ATCC 23344
Anaplasma marginale str St Maries
Frankia sp CcI3
Dehalococcoides sp BAV1
Mycoplasma genitalium G37
Mycoplasma hyopneumoniae 7448
Synechococcus sp JA 3 3Ab
Parvibaculum lavamentivorans DS 1
Lactobacillus johnsonii NCC 533
Onion yellows phytoplasma OY M
Mycoplasma mycoides subsp mycoides SC str PG1
Desulfococcus oleovorans Hxd3
Escherichia coli 536
Methylobacterium extorquens PA1
Nitrosomonas europaea ATCC 19718
Campylobacter curvus 525 92
Nitrosospira multiformis ATCC 25196
Pseudomonas aeruginosa PAO1
Burkholderia multivorans ATCC 17616
Chlamydophila abortus S26 3
Shewanella loihica PV 4
Candidatus Blochmannia pennsylvanicus str BPEN
Xylella fastidiosa 9a5c
Chlamydophila felis Fe C 56
Caulobacter crescentus CB15
Orientia tsutsugamushi Boryong
Leptospira borgpetersenii serovar Hardjo bovis JB197
Rhodopseudomonas palustris BisA53
Marinomonas sp MWYL1
Chlamydophila pneumoniae TW 183
Brucella suis 1330
Renibacterium salmoninarum ATCC 33209
Citrobacter koseri ATCC BAA 895
Mycobacterium bovis AF2122 97
Lactococcus lactis subsp lactis Il1403
Campylobacter hominis ATCC BAA 381
Polynucleobacter sp QLW P1DMWA 1
Coxiella burnetii RSA 331
Bifidobacterium longum NCC2705
Rickettsia rickettsii str Iowa
Bacillus cereus ATCC 10987
Burkholderia xenovorans LB400
Prosthecochloris vibrioformis DSM 265
Listeria monocytogenes EGD e
Burkholderia cenocepacia AU 1054
Trichodesmium erythraeum IMS101
Streptococcus thermophilus CNRZ1066
Haemophilus somnus 129PT
Bacteroides fragilis YCH46
Streptococcus pyogenes SSI 1
Shewanella sp MR 4
Chlamydophila pneumoniae CWL029
Myxococcus xanthus DK 1622
Pelobacter carbinolicus DSM 2380
Actinobacillus pleuropneumoniae serovar 3 str JL03
Rickettsia prowazekii str Madrid E
Listeria welshimeri serovar 6b str SLCC5334
Salmonella enterica subsp enterica serovar Paratyphi B str SPB7
Bartonella henselae str Houston 1
Shewanella oneidensis MR 1
Rubrobacter xylanophilus DSM 9941
Marinobacter aquaeolei VT8
Neisseria meningitidis FAM18
Acidovorax avenae subsp citrulli AAC00 1
Staphylococcus aureus subsp aureus N315
Oenococcus oeni PSU 1
Clostridium botulinum A str ATCC 19397
Streptococcus sanguinis SK36
Burkholderia pseudomallei 1710b
Bradyrhizobium sp BTAi1
Helicobacter pylori HPAG1
Staphylococcus aureus subsp aureus MW2
Pseudomonas aeruginosa PA7
Arthrobacter aurescens TC1
Candidatus Sulcia muelleri GWSS
Propionibacterium acnes KPA171202
Clostridium botulinum A str Hall
Psychrobacter cryohalolentis K5
Ralstonia solanacearum GMI1000
Polaromonas naphthalenivorans CJ2
Mycoplasma synoviae 53
Neisseria meningitidis MC58
Campylobacter jejuni subsp jejuni 81 176
Lactobacillus reuteri F275
Prochlorococcus marinus subsp marinus str CCMP1375
Rickettsia canadensis str McKiel
Lactococcus lactis subsp cremoris MG1363
Campylobacter concisus 13826
Bifidobacterium adolescentis ATCC 15703
Lactobacillus gasseri ATCC 33323
Pseudomonas putida F1
Borrelia garinii PBi
Ralstonia metallidurans CH34
Alcanivorax borkumensis SK2
Streptococcus thermophilus LMG 18311
Bacillus thuringiensis str Al Hakam
Herpetosiphon aurantiacus ATCC 23779
Azoarcus sp EbN1
Corynebacterium glutamicum R
Bacteroides thetaiotaomicron VPI 5482
Bordetella bronchiseptica RB50
Synechococcus sp JA 2 3B a 2 13
Xanthomonas oryzae pv oryzae MAFF 311018
Treponema denticola ATCC 35405
Carboxydothermus hydrogenoformans Z 2901
Campylobacter jejuni RM1221
Mycoplasma pulmonis UAB CTIP
Candidatus Protochlamydia amoebophila UWE25
Lactobacillus sakei subsp sakei 23K
Aeromonas hydrophila subsp hydrophila ATCC 7966
Shewanella sp W3 18 1
Thermus thermophilus HB8
Pseudomonas syringae pv phaseolicola 1448A
Rickettsia massiliae MTU5
Helicobacter hepaticus ATCC 51449
Mesoplasma florum L1
Mycoplasma hyopneumoniae 232
Bacillus cereus E33L
Staphylococcus aureus subsp aureus MSSA476
Azoarcus sp BH72
Silicibacter pomeroyi DSS 3
Campylobacter jejuni subsp doylei 269 97
Borrelia afzelii PKo
Ehrlichia canis str Jake
Bordetella petrii
Desulfotomaculum reducens MI 1
Gloeobacter violaceus PCC 7421
Escherichia coli O157 H7 EDL933
Staphylococcus aureus subsp aureus JH9
Nitrobacter hamburgensis X14
Rickettsia typhi str Wilmington
Novosphingobium aromaticivorans DSM 12444
Staphylococcus aureus subsp aureus USA300 TCH1516
Clostridium beijerinckii NCIMB 8052
Bacillus clausii KSM K16
Xanthomonas oryzae pv oryzae KACC10331
Clostridium botulinum F str Langeland
Lactobacillus salivarius subsp salivarius UCC118
Pediococcus pentosaceus ATCC 25745
Burkholderia mallei NCTC 10229
Francisella tularensis subsp tularensis FSC198
Symbiobacterium thermophilum IAM 14863
Pasteurella multocida subsp multocida str Pm70
Bacillus weihenstephanensis KBAB4
Sphingopyxis alaskensis RB2256
Chlamydia trachomatis A HAR 13
Solibacter usitatus Ellin6076
Delftia acidovorans SPH 1
Sinorhizobium medicae WSM419
Psychrobacter sp PRwf 1
Candidatus Pelagibacter ubique HTCC1062
Shewanella baltica OS195
Aster yellows witches broom phytoplasma AYWB
Salinispora arenicola CNS 205
Dinoroseobacter shibae DFL 12
Pelodictyon luteolum DSM 273
Arthrobacter sp FB24
Nocardioides sp JS614
Corynebacterium glutamicum ATCC 13032
Synechococcus sp WH 8102
Maricaulis maris MCS10
Methylococcus capsulatus str Bath
Clostridium botulinum A str ATCC 3502
Bacillus pumilus SAFR 032
Thiobacillus denitrificans ATCC 25259
Lactobacillus plantarum WCFS1
Thermoanaerobacter pseudethanolicus ATCC 33223
Bacillus halodurans C 125
Geobacter uraniireducens Rf4
Clostridium acetobutylicum ATCC 824
Thermotoga maritima MSB8
Francisella tularensis subsp tularensis WY96 3418
Anaeromyxobacter dehalogenans 2CP C
Thermobifida fusca YX
Erwinia carotovora subsp atroseptica SCRI1043
Yersinia pestis KIM
Leuconostoc mesenteroides subsp mesenteroides ATCC 8293
Staphylococcus aureus subsp aureus JH1
Photobacterium profundum SS9
Fervidobacterium nodosum Rt17 B1
Rhodobacter sphaeroides 2 4 1
Roseiflexus sp RS 1
Ehrlichia chaffeensis str Arkansas
Streptococcus agalactiae NEM316
Pseudomonas aeruginosa UCBPP PA14
Escherichia coli K12
Sulfurovum sp NBC37 1
Clostridium phytofermentans ISDg
Chlamydia trachomatis 434 Bu
Salmonella enterica subsp arizonae serovar 62 z4 z23
Hyphomonas neptunium ATCC 15444
Silicibacter sp TM1040
Streptococcus suis 05ZYH33
Francisella tularensis subsp holarctica OSU18
Mycobacterium ulcerans Agy99
Prochlorococcus marinus str NATL1A
Escherichia coli O157 H7 str Sakai
Mannheimia succiniciproducens MBEL55E
Chlorobium chlorochromatii CaD3
Yersinia pestis Antiqua
Shewanella sp ANA 3
Mesorhizobium loti MAFF303099
Shewanella putrefaciens CN 32
Wolbachia endosymbiont strain TRS of Brugia malayi
Mycoplasma hyopneumoniae J
Acidovorax sp JS42
Syntrophomonas wolfei subsp wolfei str Goettingen
Alkaliphilus oremlandii OhILAs
Deinococcus geothermalis DSM 11300
Pseudoalteromonas haloplanktis TAC125
Ralstonia eutropha H16
Burkholderia ambifaria AMMD
Wolbachia endosymbiont of Drosophila melanogaster
Acinetobacter baumannii ATCC 17978
Neisseria gonorrhoeae FA 1090
Herminiimonas arsenicoxydans
Clostridium perfringens str 13
Lactobacillus brevis ATCC 367
Prochlorococcus marinus str NATL2A
Shigella sonnei Ss046
Brucella melitensis 16M
Chlamydophila pneumoniae AR39
Xanthobacter autotrophicus Py2
Mycobacterium tuberculosis H37Rv
Enterobacter sakazakii ATCC BAA 894
Enterobacter sp 638
Legionella pneumophila str Lens
Rickettsia felis URRWXCal2
Staphylococcus epidermidis RP62A
Jannaschia sp CCS1
Streptomyces avermitilis MA 4680
Bradyrhizobium japonicum USDA 110
Tropheryma whipplei str Twist
Magnetococcus sp MC 1
Verminephrobacter eiseniae EF01 2
Polaromonas sp JS666
Mycobacterium smegmatis str MC2 155
Treponema pallidum subsp pallidum str Nichols
Salinibacter ruber DSM 13855
Mycoplasma pneumoniae M129
Streptococcus pyogenes M1 GAS
Francisella tularensis subsp holarctica FTA
Desulfotalea psychrophila LSv54
Escherichia coli APEC O1
Ehrlichia ruminantium str Gardel
Leptospira interrogans serovar Copenhageni str Fiocruz L1 130
Lactobacillus delbrueckii subsp bulgaricus ATCC BAA 365
Desulfovibrio desulfuricans G20
Streptococcus pneumoniae D39
Brucella canis ATCC 23365
Chromohalobacter salexigens DSM 3043
Streptococcus agalactiae A909
Haemophilus ducreyi 35000HP
Leptospira borgpetersenii serovar Hardjo bovis L550
Pseudomonas entomophila L48
Thermus thermophilus HB27
Thermotoga lettingae TMO
Salmonella enterica subsp enterica serovar Typhi str Ty2
Salmonella enterica subsp enterica serovar Choleraesuis str SC B67
Haemophilus influenzae PittGG
Bartonella bacilliformis KC583
Clostridium kluyveri DSM 555
Mycobacterium tuberculosis H37Ra
Burkholderia vietnamiensis G4
Syntrophus aciditrophicus SB
Helicobacter pylori 26695
Rhodobacter sphaeroides ATCC 17025
Mycobacterium vanbaalenii PYR 1
Candidatus Vesicomyosocius okutanii HA
Pseudoalteromonas atlantica T6c
Lactobacillus delbrueckii subsp bulgaricus ATCC 11842
Staphylococcus aureus subsp aureus str Newman
Mycoplasma agalactiae PG2
Bacillus anthracis str Ames Ancestor
Thermoanaerobacter sp X514
Staphylococcus aureus subsp aureus MRSA252
Bacillus licheniformis ATCC 14580
Salinispora tropica CNB 440
Legionella pneumophila str Paris
Arcobacter butzleri RM4018
Burkholderia mallei SAVP1
Pseudomonas stutzeri A1501
Xylella fastidiosa Temecula1
Rhodobacter sphaeroides ATCC 17029
Bdellovibrio bacteriovorus HD100
Haemophilus influenzae Rd KW20
Pseudomonas putida GB 1
Francisella tularensis subsp holarctica
Mycobacterium avium subsp paratuberculosis K 10
Rhodoferax ferrireducens T118
Clostridium difficile 630
Sodalis glossinidius str morsitans
Neisseria meningitidis Z2491
Clostridium tetani E88
Streptococcus thermophilus LMD 9
Vibrio vulnificus YJ016
Sphingomonas wittichii RW1
Staphylococcus aureus subsp aureus Mu50
Deinococcus radiodurans R1
Shigella boydii Sb227
Pseudomonas fluorescens Pf 5
Thermotoga petrophila RKU 1
Chlamydophila pneumoniae J138
Synechococcus sp RCC307
Rickettsia rickettsii str Sheila Smith
Lactobacillus acidophilus NCFM
Microcystis aeruginosa NIES 843
Desulfovibrio vulgaris subsp vulgaris str Hildenborough
Streptococcus suis 98HAH33
Burkholderia pseudomallei 1106a
Frankia sp EAN1pec
Mycobacterium tuberculosis CDC1551
Xanthomonas campestris pv vesicatoria str 85 10
Pseudomonas mendocina ymp
Chlorobium phaeobacteroides DSM 266
Brucella melitensis biovar Abortus 2308
Shigella dysenteriae Sd197
Photorhabdus luminescens subsp laumondii TTO1
Buchnera aphidicola str Sg Schizaphis graminum
Acaryochloris marina MBIC11017
Candidatus Blochmannia floridanus
Ehrlichia ruminantium str Welgevonden
Burkholderia cenocepacia HI2424
Syntrophobacter fumaroxidans MPOB
Bordetella parapertussis 12822
Salmonella typhimurium LT2
Sinorhizobium meliloti 1021
Bacteroides fragilis NCTC 9343
Rhodopseudomonas palustris BisB5
Burkholderia thailandensis E264
Gluconacetobacter diazotrophicus PAl 5
Agrobacterium tumefaciens str C58
Escherichia coli HS
Bacillus cereus subsp cytotoxis NVH 391 98
Haemophilus influenzae PittEE
Vibrio harveyi ATCC BAA 1116
Ralstonia eutropha JMP134
Petrotoga mobilis SJ95
Mycoplasma penetrans HF 2
Dechloromonas aromatica RCB
Staphylococcus aureus subsp aureus Mu3
Bartonella quintana str Toulouse
Campylobacter jejuni subsp jejuni NCTC 11168
Mycobacterium tuberculosis F11
Thermoanaerobacter tengcongensis MB4
Yersinia pestis biovar Microtus str 91001
Neorickettsia sennetsu str Miyayama
Xanthomonas axonopodis pv citri str 306
Leifsonia xyli subsp xyli str CTCB07
Desulfovibrio vulgaris subsp vulgaris DP4
Nitrobacter winogradskyi Nb 255
Methylobacillus flagellatus KT
Mycobacterium sp KMS
Staphylococcus haemolyticus JCSC1435
Pseudomonas fluorescens PfO 1
Shigella flexneri 2a str 2457T
Neisseria meningitidis 053442
Coxiella burnetii Dugway 5J108 111
Bacillus amyloliquefaciens FZB42
Chlamydia trachomatis D UW 3 CX
Roseiflexus castenholzii DSM 13941
Salmonella enterica subsp enterica serovar Paratyphi A str ATCC 9150
Staphylococcus aureus subsp aureus COL
Azorhizobium caulinodans ORS 571
Yersinia enterocolitica subsp enterocolitica 8081
Vibrio cholerae O395
Clostridium thermocellum ATCC 27405
Shigella flexneri 2a str 301
Anaeromyxobacter sp Fw109 5
Brucella ovis ATCC 25840
Paracoccus denitrificans PD1222
Pseudomonas syringae pv tomato str DC3000
Shewanella amazonensis SB2B
Bartonella tribocorum CIP 105476
Nitratiruptor sp SB155 2
Shewanella sediminis HAW EB3
Alkaliphilus metalliredigens QYMF
Bacillus cereus ATCC 14579
Burkholderia mallei NCTC 10247
Burkholderia pseudomallei 668
Staphylococcus aureus RF122
Salmonella enterica subsp enterica serovar Typhi str CT18
Staphylococcus epidermidis ATCC 12228
Geobacillus kaustophilus HTA426
Streptococcus agalactiae 2603V R
Streptococcus mutans UA159
Lactobacillus casei ATCC 334
Acinetobacter sp ADP1
Chromobacterium violaceum ATCC 12472
Chlorobium tepidum TLS
Cytophaga hutchinsonii ATCC 33406
Rickettsia bellii RML369 C
Yersinia pseudotuberculosis IP 31758
Rhizobium etli CFN 42
Helicobacter acinonychis str Sheeba
Dichelobacter nodosus VCS1703A
Janthinobacterium sp Marseille
Alkalilimnicola ehrlichei MLHE 1
Idiomarina loihiensis L2TR
Mycoplasma gallisepticum R
Yersinia pestis CO92
Oceanobacillus iheyensis HTE831
Buchnera aphidicola str Bp Baizongia pistaciae
Yersinia pestis Angola
Aeromonas salmonicida subsp salmonicida A449
Porphyromonas gingivalis W83
Granulibacter bethesdensis CGDNIH1
Frankia alni ACN14a
Dehalococcoides sp CBDB1
Mycobacterium avium 104
Listeria monocytogenes str 4b F2365
Nostoc sp PCC 7120
Chlamydia muridarum Nigg
Methylibium petroleiphilum PM1
Enterococcus faecalis V583
Vibrio vulnificus CMCP6
Acholeplasma laidlawii PG 8A
Prochlorococcus marinus str AS9601
Halorhodospira halophila SL1
Xanthomonas campestris pv campestris str 8004
Escherichia coli UTI89
Shewanella pealeana ATCC 700345
Rickettsia akari str Hartford
Caldicellulosiruptor saccharolyticus DSM 8903
Staphylococcus aureus subsp aureus NCTC 8325
Prochlorococcus marinus subsp pastoris str CCMP1986
Vibrio parahaemolyticus RIMD 2210633
Psychromonas ingrahamii 37
Legionella pneumophila str Corby
Listeria innocua Clip11262
Klebsiella pneumoniae subsp pneumoniae MGH 78578
Staphylococcus aureus subsp aureus USA300
Yersinia pestis Pestoides F
Clostridium novyi NT
Rhodopseudomonas palustris CGA009
Bacillus anthracis str Sterne
Flavobacterium johnsoniae UW101
Streptococcus pneumoniae R6
Chlamydia trachomatis L2b UCH 1 proctitis
Sulfurimonas denitrificans DSM 1251
Bacteroides vulgatus ATCC 8482
Thermosipho melanesiensis BI429
Erythrobacter litoralis HTCC2594
Chloroflexus aurantiacus J 10 fl
Ureaplasma parvum serovar 3 str ATCC 700970
Rhodospirillum rubrum ATCC 11170
Mycoplasma mobile 163K
Aquifex aeolicus VF5
Acidothermus cellulolyticus 11B
Anabaena variabilis ATCC 29413
Vibrio cholerae O1 biovar eltor str N16961
Vibrio fischeri ES114
Burkholderia sp 383
Geobacter metallireducens GS 15
Desulfitobacterium hafniense Y51
Mycobacterium sp MCS
Acidiphilium cryptum JF 5
Brucella suis ATCC 23445
Corynebacterium diphtheriae NCTC 13129
Synechococcus sp WH 7803
Mycobacterium gilvum PYR GCK
Francisella tularensis subsp novicida U112
Rhodococcus sp RHA1
Clavibacter michiganensis subsp michiganensis NCPPB 382
Bradyrhizobium sp ORS278
Corynebacterium efficiens YS 314
Mesorhizobium sp BNC1
Thermosynechococcus elongatus BP 1
Nocardia farcinica IFM 10152
Escherichia coli E24377A
Saccharophagus degradans 2 40
Legionella pneumophila subsp pneumophila str Philadelphia 1
Actinobacillus pleuropneumoniae L20
Xanthomonas campestris pv campestris str ATCC 33913
Rhizobium leguminosarum bv viciae 3841
Streptococcus pneumoniae TIGR4
Pelobacter propionicus DSM 2379
Clostridium perfringens ATCC 13124
Gammaproteobacteria
Betaproteobacteria
Alphaproteobacteria
Epsilonproteobacteria
Deltaproteobacteria
Acidobacteria
Bacteroidetes/Chlorobi
Chlamydiae/Planctomycetes
Spirochaetes
Firmicutes
Cyanobacteria
Chloroflexi
Actinobacteria
Aquificae
Thermotogae
Deinococcus/Thermus
Firmicutes
Fusobacteria

0.1
Buchnera aphidicola str Sg Schizaphis graminum
Escherichia coli O157 H7 str Sakai
Photorhabdus luminescens subsp laumondii TTO1
Aeromonas hydrophila subsp hydrophila ATCC 7966
Candidatus Ruthia magnifica str Cm Calyptogena magnifica
Saccharophagus degradans 2 40
Pasteurella multocida subsp multocida str Pm70
Wigglesworthia glossinidia endosymbiont
of Glossina brevipalpis
Aeromonas salmonicida subsp salmonicida A449
Alkalilimnicola ehrlichei MLHE 1
Psychrobacter sp PRwf 1
Sodalis glossinidius str morsitans
Candidatus Blochmannia pennsylvanicus str BPEN
Baumannia cicadellinicola str Hc
Homalodisca coagulata
Buchnera aphidicola str Bp Baizongia pistaciae
Buchnera aphidicola str Cc Cinara cedri
Acinetobacter sp ADP1
Methylococcus capsulatus str Bath
Erwinia carotovora subsp atroseptica SCRI1043
Legionella pneumophila str Corby
Xanthomonas campestris pv vesicatoria str 85 10
Francisella tularensis subsp novicida U112
Yersinia enterocolitica subsp enterocolitica 8081
Marinomonas sp MWYL1
Psychrobacter arcticus 273 4
Thiomicrospira crunogena XCL 2
Buchnera aphidicola str APS Acyrthosiphon pisum
20
26
100
100
75
100
10
93
53
100
34
76
100
95
100
100
29
32
100
9
100
100
100
100
87
100
91
100
100
22
74
100
100
93
100
65
85
46
79
99
100
100
100
99
84
66
48
5
100
75
100
46
73
A.
0.07
Sodalis glossinidius str. 'morsitans'
Thiomicrospira crunogena XCL-2
Acinetobacter sp. ADP1
Alkalilimnicola ehrlichei MLHE-1
Xanthomonas campestris pv. vesicatoria str. 85-10
Methylococcus capsulatus str. Bath
Candidatus Ruthia magnifica str. Cm Calyptogena magnifica
Erwinia carotovora subsp. atroseptica SCRI1043
Buchnera aphidicola str. Cc Cinara ce
Francisella tularensis subsp. novicida U112
Psychrobacter sp. PRwf-1
Escherichia coli O157H7 str. Sakai
Saccharophagus degradans 2-40
Photorhabdus luminescens subsp. laumondii TTO1
Aeromonas hydrophila subsp. hydrophila ATCC 7966
Marinomonas sp. MWYL1
Baumannia cicadellinicola str. Hc Homalodisca c
Buchnera aphidicola str. APS Acyrth
Wigglesworthia glossinidia endosymbiont of Glo
Buchnera aphidicola str. Bp Baizon
Candidatus Blochmannia pennsylvanicus str. BP
Buchnera aphidicola str. Sg Schiz
Pasteurella multocida subsp. multocida str
Yersinia enterocolitica subsp. enterocolitica 8081
Legionella pneumophila str. Corby
Aeromonas salmonicida subsp. salmonicida A449
Psychrobacter arcticus 273-4
54
37
100
14
10
28
50
100
48
25
45
99
98
100
100
52
37
100
83
100
10
43
24
100
100
8
94
86
100
75
100
37
45
56
31
49
100
64
22
94
23
38
7678
45
22
14
100
72
4
66
2
B.
Proteins rRNA

ORF00955
gi73748443
gi57234592
gi86609871
gi86605128
gi17229086
gi75910405
gi113476515
gi16329957
gi56752516
gi81300331
gi33862041
gi78779962
gi33865147
gi78213583
gi78184182
gi113953748
gi33863774
gi72382854
gi33241089
gi22298184
gi37521852
ZAVAM73TF
Thermomicrobium roseum DSM 5159
Dehalococcoides sp. CBDB1
Dehalococcoides ethenogenes 195
Synechococcus sp. JA 2 3Bʼa 2 13
Synechococcus sp. JA 3 3Ab
Nostoc sp. PCC 7120
Anabaena variabilis ATCC 29413
Trichodesmium erythraeum IMS101
Synechocystis sp. PCC 6803
Prochlorococcus marinus subsp. pastoris str. CCMP1986
Prochlorococcus marinus str. MIT 9312
Synechococcus sp. WH 8102
Synechococcus sp. CC9605
Prochlorococcus marinus str. MIT 9313
Prochlorococcus marinus str. NATL2A
Prochlorococcus marinus subsp. marinus str. CCMP1375
Thermosynechococcus elongatus BP 1
Gloeobacter violaceus PCC 7421

Search with HMMs
!54

Search with HMMs
!55

What Else Needed for This to Be Better?

What Else Needed for This to Be Better?
• More Phylogenetically Diverse Genomes
• More Markers esp. Taxon Specific Markers
• Better / Faster Searching
• Longer reads
• Many sequences at once
• Many genes at once
• Alpha and Beta Diversity
• Automated Masking

Zorro - Automated Masking
cetoTrueTree
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
200 400 800 1600 3200
DistancetoTrueTree
Sequence Length
200
no masking
zorro
gblocks
200
Wu M, Chatterji S, Eisen JA (2012) Accounting For Alignment
Uncertainty in Phylogenomics. PLoS ONE 7(1): e30288. doi:
10.1371/journal.pone.0030288

Representative
Genomes
Extract
Protein
Annotation
All v. All
BLAST
Homology
Clustering
(MCL)
SFams
Align &
Build
HMMs
HMMs
Screen for
Homologs
New
Genomes
Extract
Protein
Annotation
Figure 1
Sharpton et al. 2013
A
B
C
Improving II: More Families

Kembel Combiner
typically used as a qualitative measure because duplicate s
quences are usually removed from the tree. However, the
test may be used in a semiquantitative manner if all clone
even those with identical or near-identical sequences, are i
cluded in the tree (13).
Here we describe a quantitative version of UniFrac that w
call “weighted UniFrac.” We show that weighted UniFrac b
haves similarly to the FST test in situations where both a
FIG. 1. Calculation of the unweighted and the weighted UniFr
measures. Squares and circles represent sequences from two differe
environments. (a) In unweighted UniFrac, the distance between t
circle and square communities is calculated as the fraction of t
branch length that has descendants from either the square or the circ
environment (black) but not both (gray). (b) In weighted UniFra
branch lengths are weighted by the relative abundance of sequences
the square and circle communities; square sequences are weight
twice as much as circle sequences because there are twice as many tot
circle sequences in the data set. The width of branches is proportion
to the degree to which each branch is weighted in the calculations, an
gray branches have no weight. Branches 1 and 2 have heavy weigh
since the descendants are biased toward the square and circles, respe
tively. Branch 3 contributes no value since it has an equal contributio
from circle and square sequences after normalization.
Kembel SW, Eisen JA, Pollard KS, Green JL (2011) The Phylogenetic Diversity of
Metagenomes. PLoS ONE 6(8): e23214. doi:10.1371/journal.pone.0023214

Phylosift - Mining the Global Metagenome
Jonathan
Eisen
Students and other staff:
- Eric Lowe, John Zhang, David Coil
!
Open source community:
- BLAST, LAST, HMMER, Infernal,
pplacer, Krona, metAMOS, Bioperl,
Bio::Phylo, JSON, etc. etc.
!
PhyloSift is open source software:
-http://phylosift.wordpress.org
-http://github.com/gjospin/phylosift
Erick Matsen
FHCRC
Todd Treangen
BNBI, NBACC
Holly
Bik
Tiffanie
Nelson
Mark
Brown
Aaron
Darling
Guillaume
Jospin
Supported by DHS Grant

Output 1: Taxonomy
Taxonomic
summary
plots in
Krona
(Ondov et al
2011)

Tree Reconciliation in PhyloSift

Tree Reconciliation in PhyloSift
Environmental
Sequences
Named
Taxa

Output 2: Phylogenetic Tree of Reads
Placement tree from 2 week old infant gut data

Edge PCA: Identify lineages
that explain most variation
among samples
Matsen and Evans 2013, Darling et al Submitted.
Output: Edge PCA

QIIME and Edge PCA on 110
fecal metagenomes from
Yatsunenko et al 2012 Nature. 
 
Sequenced with 454, to about
150Mbp/metagenome
Darling et al
Submitted.
Edge PCA vs. UNIFRAC PCA
Edge PCA: Matsen
and Evans 2013

Output 3: Edge PCA
" Edge PCA for exploratory data analysis (Matsen and Evans 2013)
" Given E edges and S samples:
− For each edge, calculate difference in placement mass on either side of edge
− Results in E x S matrix
− Calculate E x E covariance matrix
− Calculate eigenvectors, eigenvalues of covariance matrix
" Eigenvector: each value indicates how “important” an edge is in explaining differences
among the S samples
Example calculating a matrix entry for an edge:
This edge gets 5-2=3
mass=5 mass=2

Phylosift/ pplacer Workflow
Aaron Darling, Guillaume Jospin, Holly Bik, Erik Matsen, Eric Lowe,
and others
Input Sequences
rRNA workflow
protein workflow
profile HMMs used to align
candidates to reference alignment
Taxonomic
Summaries
parallel option
hmmalign
multiple alignment
LAST
fast candidate search
pplacer
phylogenetic placement
LAST
LAST
search input against references
hmmalign
multiple alignment
hmmalign
multiple alignment
Infernal
multiple alignment
LAST
<600 bp
>600 bp
Sample Analysis &
Comparison
Krona plots,
Number of reads placed
for each marker gene
Edge PCA,
Tree visualization,
Bayes factor tests
eachinputsequencescannedagainstbothworkflows

Phylosift Markers
• PMPROK – Dongying Wu’s Bac/Arch
markers
• Eukaryotic Orthologs – Parfrey 2011 paper
• 16S/18S rRNA
• Mitochondria - protein-coding genes
• Viral Markers – Markov clustering on
genomes
• Codon Subtrees – finer scale taxonomy
• Extended Markers – plastids, gene families

More Markers
Phylogenetic
group
Genome
Number
Gene
Number
Maker
Candidate
Archaea 62 145415 106
Actinobacteria 63 267783 136
Alphaproteoba 94 347287 121
Betaproteobac 56 266362 311
Gammaproteo 126 483632 118
Deltaproteoba 25 102115 206
Epislonproteo 18 33416 455
Bacteriodes 25 71531 286
Chlamydae 13 13823 560
Chloroflexi 10 33577 323
Cyanobacteria 36 124080 590
Firmicutes 106 312309 87
Spirochaetes 18 38832 176
Thermi 5 14160 974
Thermotogae 9 17037 684
Wu D, Jospin G, Eisen JA (2013) Systematic Identification of Gene
Families for Use as “Markers” for Phylogenetic and Phylogeny-Driven
Ecological Studies of Bacteria and Archaea and Their Major
Subgroups. PLoS ONE 8(10): e77033. doi:10.1371/journal.pone.
0077033

Better Reference Tree
Lang JM, Darling AE, Eisen JA (2013)
Phylogeny of Bacterial and Archaeal
Genomes Using Conserved Genes:
Supertrees and Supermatrices. PLoS
ONE 8(4): e62510. doi:10.1371/
journal.pone.0062510

Stalking the Fourth Domain in Metagenomic Data:
Searching for, Discovering, and Interpreting Novel, Deep
Branches in Marker Gene Phylogenetic Trees
Dongying Wu1
, Martin Wu1,4
, Aaron Halpern2,3
, Douglas B. Rusch2,3
, Shibu Yooseph2,3
, Marvin Frazier2,3
,
J. Craig Venter2,3
, Jonathan A. Eisen1
*
1 Department of Evolution and Ecology, Department of Medical Microbiology and Immunology, University of California Davis Genome Center, University of California
Davis, Davis, California, United States of America, 2 The J. Craig Venter Institute, Rockville, Maryland, United States of America, 3 The J. Craig Venter Institute, La Jolla,
California, United States of America, 4 University of Virginia, Charlottesville, Virginia, United States of America
Abstract
Background: Most of our knowledge about the ancient evolutionary history of organisms has been derived from data
associated with specific known organisms (i.e., organisms that we can study directly such as plants, metazoans, and
culturable microbes). Recently, however, a new source of data for such studies has arrived: DNA sequence data generated
directly from environmental samples. Such metagenomic data has enormous potential in a variety of areas including, as we
argue here, in studies of very early events in the evolution of gene families and of species.
Methodology/Principal Findings: We designed and implemented new methods for analyzing metagenomic data and used
them to search the Global Ocean Sampling (GOS) Expedition data set for novel lineages in three gene families commonly
used in phylogenetic studies of known and unknown organisms: small subunit rRNA and the recA and rpoB superfamilies.
Though the methods available could not accurately identify very deeply branched ss-rRNAs (largely due to difficulties in
making robust sequence alignments for novel rRNA fragments), our analysis revealed the existence of multiple novel
branches in the recA and rpoB gene families. Analysis of available sequence data likely from the same genomes as these
novel recA and rpoB homologs was then used to further characterize the possible organismal source of the novel sequences.
Conclusions/Significance: Of the novel recA and rpoB homologs identified in the metagenomic data, some likely come from
uncharacterized viruses while others may represent ancient paralogs not yet seen in any cultured organism. A third
possibility is that some come from novel cellular lineages that are only distantly related to any organisms for which
sequence data is currently available. If there exist any major, but so-far-undiscovered, deeply branching lineages in the tree
of life, we suggest that methods such as those described herein currently offer the best way to search for them.
Citation: Wu D, Wu M, Halpern A, Rusch DB, Yooseph S, et al. (2011) Stalking the Fourth Domain in Metagenomic Data: Searching for, Discovering, and
Interpreting Novel, Deep Branches in Marker Gene Phylogenetic Trees. PLoS ONE 6(3): e18011. doi:10.1371/journal.pone.0018011
Editor: Robert Fleischer, Smithsonian Institution National Zoological Park, United States of America
Received October 25, 2010; Accepted February 20, 2011; Published March 18, 2011
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: The development and main work on this project was supported by the National Science Foundation via an ‘‘Assembling the Tree of Life’’ grant
(number 0228651) to to Jonathan A. Eisen and Naomi Ward. The final work on this project was funded by the Gordon and Betty Moore Foundation (through

Figure 1. Phylogenetic tree of the RecA superfamily. All RecA sequences were grouped into clusters using the Lek algorithm. Representatives
of each cluster that contained .2 members were then selected and aligned using MUSCLE. A phylogenetic tree was built by from this alignment
using PHYML; bootstrap values are based on 100 replicas. The Lek cluster ID precedes each sequence accession ID. Proposed subfamilies in the RecA
superfamily are shaded and given a name on the right. Five of the proposed subfamilies contained only GOS sequences at the time of our initial
analysis (RecA-like SAR, Phage SAR1, Phage SAR2, Unknown 1 and Unknown 2) and are highlighted by colored shading. As noted on the tree and in
the text, sequences from two Archaea that were released after our initial analysis group in the Unknown 2 subfamily.
doi:10.1371/journal.pone.0018011.g001
Stalking the Fourth Domain
PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e18011

Five RecA subfamilies were identified as being novel (i.e., only seen in metagenomic data) in our initial analyses. GOS metagenome assemblies that encode members of
these subfamilies were identified and the genes neighboring the novel RecAs were characterized. The neighboring gene descriptions are based on the top BLASTP hits
against the NRAA database; taxonomy assignments are based on their closest neighbor in phylogenetic trees built from the top NRAA BLASTP hits.
doi:10.1371/journal.pone.0018011.t002
Figure 2. The largest assembly from the GOS data that encodes a novel RecA subfamily member (a representative of subfamily
Unknown 2). This GOS assembly (ID 1096627390330) encodes 33 annotated genes plus 16 hypothetical proteins, including several with similarity to
known archaeal genes (e.g., DNA primase, translation initiation factor 2, Table 2). The arrow indicates a novel recA homolog from the Unknown 2
subfamily (cluster ID 9).
PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e18011

Methods
Identification of deeply-branching ss-rRNA sequences
these 340 sequences were extracted from the European Ribosomal
RNA database [66] and then manually curated to remove
columns with more than 90% gaps or with poor alignment
Figure 3. Phylogenetic tree of the RpoB superfamily. All RpoB sequences were grouped into clusters using the Lek algorithm. Representatives
of each cluster that contained .2 members were then selected and aligned using MUSCLE. A phylogenetic tree was built by from this alignment
using PHYML; bootstrap values are based on 100 replicas. The Lek cluster ID precedes each sequence accession ID. Proposed subfamilies in the RpoB
superfamily are shaded and given a name on the right. The two novel RpoB clades that contain only GOS sequences are highlighted by the colored
panels.
Stalking the Fourth Domain

UC Davis EVE161 Lecture 16 by @phylogenomics

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