Introduction to Microbial Phylogenomics Course EVE161

Lecture 1:

EVE 161: 
Microbial Phylogenomics
!

Lecture #1:
Introduction
!
UC Davis, Winter 2014
Instructor: Jonathan Eisen

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

!1

Where we are going and where we have been

• Previous lecture:
!

• Current Lecture:
! 1. Introduction
• Next Lecture:
! 2. Evolution of DNA sequencing


!2

Lecture 1 Outline

• Course details
• Four eras of sequencing
• Introduction to phylogeny


!3

Main topics of the course

• DNA sequence based studies of microbial
diversity
• Four Eras of sequencing
! The Tree of Life
! rRNA from environments
! Genome Sequencing
! Metagenomics


!4

Textbook/Reading

• Each lecture will have some associated
background reading and 1+ primary
literature papers
• Whenever possible, the primary literature
used will be “Open Access” material
• There will also be news stories, blogs and
other “media” to review / read


!5

What you should learn from the course

• History of sequence based studies of
microbial diversity
• Current practice in sequence based
studies of microbial diversity
• Broad view of what we know about
microbial diversity
• How to read and analyze a research paper


!6

Grading

•
•
•
•
•

Attendance and class participation 20 %
Weekly assignments 20 %
Midterm 20 %
Final presentation 20%
Final exam 20%


!7

Student project

• Select 1-2 papers on one of the topics of the
course (approval needed)
• Review the paper and write up a summary of
your assessment of the paper (more detail on
this later)
• Post your assessment on the course blog
• Present a short summary of what you did to the
class
• Ask and answer questions about your and other
people’s reviews on the course blog


!8

Contact information

• Jonathan Eisen, Professor
– jaeisen@ucdavis.edu
– Phone 752-3498
– Office Hours: TBD

• Holly Ganz
– hhganz@ucdavis.edu
– Office Hours: TBD


!9

Course Information
• SmartSite
• Also will be posting for the broader community at http://
microbe.net/eve161


!10

Introduction to EVE161


!11

Microbial Diversity


!12

Microbial Diversity

• Microbes are small
• But diversity and numbers are
very high
• Appearance not a good
indicator of type or function
• Field observations of limited
value


!13

Diversity of Form


!14

Diversity of Function
The Bad

The Good

The Consumable

The Burnable

The Unusual

The Planet


!15

Phylogenetic Diversity


!16

Phylogeny was central to Darwin’s Work on Natural Selection


!17

Phylogeny
• Phylogeny is a description of the evolutionary history of
relationships among organisms (or their parts).
• This is portrayed in a diagram called a phylogenetic
tree.
• Phylogenetic trees are used to depict the evolutionary
history of populations, species and genes.
• The Tree of Life refers to the concept that all living
organisms are related to one another through shared
ancestry.

Ch. 25.1

!18

Four Eras of Sequence & Microbial Diversity


!19

Relevant Reading
• Eisen JA. Environmental shotgun sequencing: its
potential and challenges for studying the hidden world of
microbes. PLOS Biology 5(3): e82.


!20

Moore’s Law


!21

Era I: rRNA Tree of Life

Era I:
rRNA Tree of Life


!22

Era I: rRNA Tree of Life

Era I:
rRNA Tree of Life
Lectures 3-4

!23

Ernst Haeckel 1866

Plantae
Protista
Animalia

!24

Whittaker – Five Kingdoms 1969

Monera
Protista
Plantae
Fungi
Animalia

!25

Carl Woese

http://mcb.illinois.edu/faculty/
proﬁle/1204


!26


!27


!28


!29

Woese and Fox  

• Abstract: A phylogenetic analysis based upon ribosomal
RNA sequence characterization reveals that living
systems represent one of three aboriginal lines of
descent: (i) the eubacteria, comprising all typical bacteria;
(ii) the archaebacteria, containing methanogenic bacteria;
and (iii) the urkaryotes, now represented in the
cytoplasmic component of eukaryotic cells.


!30

Propose “three aboriginal lines of descent”
! Eubacteria
! Archaebacteria
! Urkaryotes


!31

Woese 1987


!32

• Appearance of
microbes not
informative (enough)
• rRNA Tree of Life
identified two major
groups of organisms  
w/o nuclei
• rRNA powerful for
many reasons, though
not perfect


!33

Tree of Life

• Three main kinds of organisms
! Bacteria
! Archaea
! Eukaryotes
• Viruses not alive, but some call them microbes
• Many misclassifications occurred before the use of
molecular methods


!34

Tree of Life

adapted from Baldauf, et al., in Assembling the Tree of Life, 2004


!35

Most of the phylogenetic diversity of life is microbial

adapted from Baldauf, et al., in Assembling the Tree of Life, 2004


!36

Simpliﬁed, Rooted Tree of Life


!37

Alternative rooted tree of life

Archaea
Archaea


!38

Era II: rRNA in the Environment

Era II:
rRNA in the Environment


!39

Era II: rRNA in the Environment

Era II:
rRNA in the Environment
Lectures 5-9

!40

Plant/Animal Field Studies


!41

Microbial Field Studies


!42

Culturing Microbes


!43

Great Plate Count Anomaly


!44


Culturing

Microscopy


!45


Culturing

Count

Microscopy

Count


!46


Culturing

Count

Microscopy

<<<<

Count


!47

Problem because
appearance not
effective for “who
is out there?” or
“what are they
doing?”

Culturing

Count

Microscopy

<<<<

Count


!48

Solution?

Problem because
appearance not
is out there?” or
“what are they
doing?”

Culturing

Count

Microscopy

<<<<

Count


!49

Solution?

Problem because
appearance not
is out there?” or
“what are they
doing?”

DNA

Culturing

Count

Microscopy

<<<<

Count


!50

Analysis of uncultured microbes

Collect from
environment

!51

PCR and phylogenetic analysis of rRNA genes
DNA
extraction

PCR

PCR

Phylogenetic tree
rRNA1

Sequence alignment = Data matrix

Yeast

A

C

A

C

A

T

A

C

A G

T

A G A

C

T

A G

rRNA1
5’ ...TACAGTATAGG
TGGAGCTAGCGAT
CGATCGA... 3’

C

E. coli
Humans

rRNA1
Yeast

E. coli

Sequence
rRNA genes

Makes lots
of copies of
the rRNA
genes in
sample

Humans

A

T

A G
T


!52

DNA
extraction

PCR

PCR

Phylogenetic tree
rRNA1


rRNA2

Yeast

A

C

A

C

A

T

A

C

A G

T

A G A

C

Humans

T

A

T

A G

T

Yeast

T

A

C

A G

rRNA1
5’ ...ACACACATAG
GTGGAGCTAGCGA
TCGATCGA... 3’

C

E. coli
Humans

rRNA1
rRNA2

E. coli

Sequence
rRNA genes

Makes lots
of copies of
the rRNA
genes in
sample

T

A G

rRNA2
5’ ...TACAGTATAGG
TGGAGCTAGCGAT
CGATCGA... 3’


!53

DNA
extraction

PCR

PCR

Phylogenetic tree
rRNA1


rRNA2

rRNA1

A

C

A

C

rRNA2

T

A

C

A G

T

C

A

C

T

G

T

rRNA4

C

A

C

A G

T

E. coli

A G A

C

T

A

T

A G

T

Yeast

Yeast

C

Humans

Humans

E. coli

A

rRNA3

rRNA4

rRNA3

Sequence
rRNA genes

Makes lots
of copies of
the rRNA
genes in
sample

T

A

C

A G

rRNA1
5’...ACACACATAGGTGGAGC
TAGCGATCGATCGA... 3’
rRNA2
5’..TACAGTATAGGTGGAGCT
AGCGACGATCGA... 3’

T

A G

rRNA3
5’...ACGGCAAAATAGGTGGA
TTCTAGCGATATAGA... 3’
rRNA4
5’...ACGGCCCGATAGGTGG
ATTCTAGCGCCATAGA... 3’


!54


PCR


!55

Major phyla of bacteria & archaea (as of 2002)

No cultures
Some cultures

!56

The Hidden Majority

Hugenholtz 2002

Richness estimates

Bohannan and Hughes 2003


!57

Human microbiome case study

Censored

Censored


!58

Built Environment Case Study
Microbial Biogeography of Public Restroom Surfaces
Gilberto E. Flores1, Scott T. Bates1, Dan Knights2, Christian L. Lauber1, Jesse Stombaugh3, Rob Knight3,4,
Noah Fierer1,5*
Bacteria of Public Restrooms
1 Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Computer Science,
University of Colorado, Boulder, Colorado, United States of America, 3 Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, United
States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Department of Ecology and Evolutionary
Biology, University of Colorado, Boulder, Colorado, United States of America

Abstract

We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the
diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited
by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of
the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla:
www.nature.com/ismej
Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those
found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with
Figure hands. On toilet surfaces, gut-associated taxa wereof discriminating suggesting fecal contamination of theseblue indicates low
3. Cartoon illustrations of the relative abundance more prevalent, taxa on public restroom surfaces. Light surfaces. Floor
abundance while dark blue indicates high abundance of taxa. (A) Although skin-associated taxa (Propionibacteriaceae, Corynebacteriaceae,
surfaces were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated
Staphylococcaceae and Streptococcaceae) were abundant on all surfaces, they were relatively more abundant on surfaces routinely touched with
bacteria, especially the Propionibacteriaceae, dominated surfaces routinely touched Prevotellaceae and Bacteroidaceae) were most
hands. (B) Gut-associated taxa (Clostridiales, Clostridiales group XI, Ruminococcaceae, Lachnospiraceae, with our hands. Certain taxa were more
common surfaces. (C) Although soil-associated taxa (Rhodobacteraceae, Rhizobiales, Microbacteriaceae and Nocardioidaceae) were in low
abundant on toilet in female than in male restrooms as vagina-associated Lactobacillaceae were widely distributed in female
restrooms, likely from urine were relatively more abundant SourceTracker algorithm confirmed Figure not drawn to scale.
abundance on all restroom surfaces, they contamination. Use of the on the floor of the restrooms we surveyed.many of our taxonomic
doi:10.1371/journal.pone.0028132.g003 was the primary source of bacteria on restroom surfaces. Overall, these results demonstrate that
observations as human skin
restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with Bacteria of P
clear
1
1
1,2
1,2
1,2
linkages between communities on or in different body sites and those communities found on restroom surfaces. More
Steven W Kembel , Evan Jones , Jeff Kline , Dale Northcutt , Jason Stenson ,
Results of SourceTracker microbes are commonly found
the stall in), they were work is relevant to the public women field as we show that human-associatedanalysis support the taxonomic
likely dispersed manually after health used
1
1
1,2
1,3
generally, this
Bohannan , G Z Brown and Jessica L Green
Ann
time, the M Womack , Brendan JM 100
SOURCES
on Coupling
1
Bathroomthe toilet.restroom surfaces suggesting thatwith those of the could readily be transmitted between individuals by the touching
biogeography. Bythese observations bacterial pathogens patterns highlighted above, indicating that human skin was the
Biology and the Built Environment Center, Institute of Ecology and Evolution, Department of
distribution of gut-associated bacteriademonstrate that we can of high-throughput analyses of bacterial communities to determine
indicate that routine use use primary source of bacteria on all public restroom surfaces
Soil
un to take
of surfaces in
2
swabbing differentsurfaces. Furthermore, we and fecal-associated bacteria
Biology, University of Oregon, Eugene, OR, USA; Energy Studies in Buildings Laboratory,
toilets results in the bacteria onurine- surfaces, an approach which examined, used to track pathogen transmission and teston or
sources of dispersal of indoor
could be while the human gut was an important source the
Water
80
of outside
Department of Architecture, University of Oregon, Eugene, OR, USA and 3Santa Fe Institute,
public restrooms, researchers While these results are not unexpected,
around the toilet, and urine was an important source in women’s
throughout the of hygiene practices.
restroom.
efficacy
Mouth
Santa
om plants Fe, NM, USA
restrooms (Figure 4, Table S4). Contrary to expectations (see
they do highlight the importance of hand-hygiene when using
determined that microbes vary in
Urine
60
SourceTracker 6(11): e28132.
ours after
where theypublic restrooms since these Knights D, Lauber CL,also be potential(2011) above), soil was not identified by theSurfaces. PLoS ONE algorithm as
come from Flores GE, Bates ST, surfaces could Stombaugh J, et al.
Citation: dependMicrobial Biogeography of Public Restroom
Gut
being a major source of bacteria on any of the surfaces, including
vehicles for the transmission of human pathogens. Unfortunately,
doi:10.1371/journal.pone.0028132
ing on the surface (chart). have documented that college students (who are
ere shut
floors (Figure 4). Although the floor samples contained family-level
previous studies R. Liles, Auburn University, United States of America
Buildings are complex ecosystems that house trillions of microorganisms interactingSkin each
with
Editor: Mark
40
taxa that are common in soil, the SourceTracker algorithm
likely the most frequent users of the studied restrooms) are not
ortion of
other, with humans and with their environment. Understanding the ecological and evolutionary
Received September 12, 2011; Accepted November 1, 2011; Published November 23, 2011
processes that determine the diversity and composition of the built environment microbiome—thepant in indoor microbial of hand-washers [42,43].
probably underestimates the relative importance of sources, like
always the most diligent
e human
Copyright: ß 2011 Flores et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
community of microorganisms that live indoors—is important for understanding the relationship
20
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The ISME Journal (2012), 1–11
& 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12

ORIGINAL ARTICLE

between building design, biodiversity and human health. In this study, we used high-throughputecology research, Peccia
Funding: This
was
sequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes and
thinks that the field workand the Howard with funding from the Alfred P. Sloan had no role andstudy design, data collection and analysis, in part bytothe National
has supported Hughes Medical Institute. The funders Foundation in their Indoor Environment program, and decision publish, or
Institutes of Health
airborne bacterial communities at 0 health-care facility. We quantified airborne bacterial community
a
preparation
structure and environmental conditions in patient rooms exposed to mechanical or windowyet to gel. And the of the manuscript.
wh i c h
Sloan
Competing Interests: The authors have declared that no competing interests exist.
ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities was
26 JanuOlsiewski
lower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbialFoundation’sE-mail: noah.fierer@colorado.edu
*
communities than did window-ventilated rooms. Bacterial communities in indoor environmentsshares some of his conJournal,
contained many taxa that are absent or rare outdoors, including taxa closely related to potential
hanically
human pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relativecern. “Everybody’s gencommunities and revealed a greater diversity of bacteria on
Introduction
humidity and temperature, were correlated with the diversity and composition of indoor bacterialerating vast amounts of
had lower
indoor surfaces than captured using cultivation-based techniques
communities. The relative abundance of bacteria closely related to human pathogens was higher
More data sets
y than ones with openthan outdoors, and higher in rooms withquantify those con- lower relative humidity. looking across than ever, individuals across the globe spend a large [10–13]. Most of the organisms identified in these studies are
they move around. But to lower airflow rates and data,” she says, but
indoors winportion of their
The observed relationship between building design and airborne bacterial can be difficult because groups choose lives indoors, yet relatively little is known about the
ility of fresh air translated tributions, Peccia’s team has had to develop diversity suggests that
dif- indoor environments. Of the studies that related to human commensalsrestroom surfaces. Communities are
Figure 2. Relationship between bacterial communities associated with ten public suggesting that the organisms wer
microbial diversity of of the unweighted UniFrac distance
we can manage indoor environments, altering through building design and operation the community
growing
the surfaces but rather were
PCoA
rtions of microbes associ- new methods to collect airborne bacteria and our timeanalytical tools. With have examined microorganisms associated with indoormatrix. Each point represents touching) on indirectly (e.g. shedding of skindeposited(a
Sloan support, clusters distinct from surfaces touchedenviron- not actively a single sample. Note that the floor (triangles) and toilet
of microbial species that potentially colonize the human microbiome during ferent indoors.
form
with hands. directly (i.e.
or
cells) by
The ISME Journal extract their DNA, 26 January 2012; doi:10.1038/ismej.2011.211
ments, most have relied upon cultivation-based techniques to
doi:10.1371/journal.pone.0028132.g002
an body, and consequently, advance online publication,as the microbes are much though, a data archive and integrated analythumans. Despite these efforts, we still have an incomplete
Subject Category: microbial population and community ecology
detect organisms residing on a variety of household surfaces [1–5].
understanding of bacterial communities associated with indoor
pathogens. Although this less abundant in builtthan on surfaces.
ical tools dispersal;
Keywords: aeromicrobiology; bacteria; air environment microbiome; community ecology; are in the works. Not surprisingly, these studies have identified surfaces in kitchens

g on February 9, 2012

Do
or
Do in
or
ou
t
St
all
in
Fa Sta
uc
et ll ou
So han t
ap
d
dis les
pe
ns
To
T
e
ile oile r
tf
lus t sea
hh t
a
To ndle
ile
tf
lo
Si or
nk
flo
or

ck to pre-

Average contribution (%)

Architectural design influences the diversity and
structure of the built environment microbiome

high diversity of floor communities is likely due to the frequency of because limitations of traditional 16abundances of s
environments related differences in the relative S rRNA gene

environmental filtering In one recent study, they used air filters
hat having natural airflow
To foster collaborations between micro- with the bottombacterial which would track in a diversity sequencing techniques have made replicate sampling
and restrooms as being hot spots of of shoes, contamination.
contact
some surfaces (Figure 1B, Table S2). Most notably
cloning and
Because several of microorganisms from a variety ofto survive on
pathogenic bacteria are known sources including soil, which is characterizations of the communities prohibitive.
Green says answering that to sample airborne particles and microbes biologists, architects, and building scientists,
and in-depth were clearly more abundant on certain surfaces
surfaces for extended to be a time [6–8], these studies are of
known
microbial
With the advent of high-throughput sequencing techniques, we
Introduction
microbiome—includes the foundation and comclinical data; she’s hoping in a classroom during 4 days during which human pathogensalso sponsored a symposiuminperiods of highly-diverse of human habitat [27,39]. Indeed, restrooms than male restrooms (Figure 1B). Some
obvious importance
preventing the spread
disease.
bacteria commonly associated with soil (e.g. Rhodobacteraceae, investigate are the most common, and often most abu
family indoor microbial communities at an
can now
mensals interacting
with
ital to participate in a study 90% of theirwere present and 4 days during with each other and haveof the built environment widely recognized that the majority of on average, depth andthe vagina understand reproductive age
on the microbiome their
However, it is Rhizobiales, Microbacteriaceae and Nocardioidaceae) were, unprecedented found in begin to of healthy the relationship
now
Humans spend up to students lives indoors
environment (Eames et al., 2009). There
been
(Klepeis et al., 2001). Consequently,roomway we
the Slides few attempts to comprehensively survey the built Taught by Jonathanon floor surfaces (Figure 2014 S2).
for UC Davis EVE161Indoor Air conference in Austin, abundant cultivated [9]Winter 3C, between humans,and are relatively built abundant in male urine
Course microorganisms cannot be readily Eisen and thus, the Table
more
microbes and the less environment. !59
dence of hospital-acquired which the
was vacant. They measured at the 2011

design and operate the indoor environment has a

overall diversityInterestingly, some of the toilet flush handles harbored bacterial
of microorganisms associated with indoor

analysis of female urine samples collected as par

Era III: Genome Sequencing

Era III:
Genome Sequencing


!60

Era III: Genome Sequencing

Era III:
Genome Sequencing
Lectures 10-14

!61

1st Genome Sequence


Fleischmann
et al. 1995 !62

Genomes 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


!63


!64

Metabolic Predictions


!65

Lateral Gene Transfer

Perna et al. 2003

!66

Network of Life
Bacteria

Archaea

Eukaryotes
Figure from Barton, Eisen et al.
“Evolution”, CSHL Press.
Based on tree from Pace NR, 2003.

!67

GEBA Case Study
• Phylogenetic diversity poorly
sampled
• GEBA project at DOE-JGI
correcting this


!68

Era IV: Genomes in the environment

Era IV:
Genomes in the Environment


!69

Era IV: Genomes in the environment

Era IV:
Genomes in the Environment
Lectures 15-19

!70

Era IV: Metagenomics
DNA
extraction
Sequence
all genes

PCR

5’ ...TACAGTATAGGTG
GAGCTAGCGATCGAT
CGA... 3’


Delong GENOMIC FRAGMENTS FROM PLANKTONIC MARINE ARCHAEA
Lab

593

ments isolated from fosmid clones
with various restriction endonucle10 kb, the F-factor-based vector
the fosmid subfragments. Partial
of restriction enzyme to 1 ⇥g of
mixture. The reaction mixture was
removed at 10, 40, and 60 min.
dding 1 ⇥l of 0.5 M EDTA to the
e. The partially digested DNA was
s described above except using a 1he sizes of the separated fragments
n standards. The distances of the
d SP6 promoter sites on the excised
pmol of T7- or SP6-speciﬁc oligol) and hybridizing with Southern

artial sequences reported in Table
the following accession numbers:
U40243, U40244, and U40245. The
and EF2 have been submitted to
and U41261.

FIG. 1. Flowchart depicting the construction and screening of an environmental library from a mixed picoplankton sample. MW, molecular weight;
PFGE, pulsed-ﬁeld gel electrophoresis.


D ownloa de d from jb.a sm .org a t U N IV O

fosmid and pBAC clones digested
probed with labeled T7 and SP6
eled subclones and PCR fragments
otgun sequencing described above.
e estimates from the partial digesof the fosmids and their subclones.
and DeSoete distance (9) analyses
n using GDE 2.2 and Treetool 1.0,
(RDP) (23). DeSoete least squares
ng pairwise evolutionary distances,
to account for empirical base fretained from the RDP, version 4.0
rRNA sequences were performed
the RDP. For distance analyses of
lutionary distances were estimated
d tree topology was inferred by the
n addition and global branch swapprotein sequences, the Phylip proaddition and ordinary parsimony

!72

D ownloa de d from w

generated
eorhodopresence of
ndwidth is
absorption
. The rednm in the
ated Schiff
ably to the

Delong Lab

on was des in a cell
ward transin proteornd only in
(Fig. 4A).
edium was
ce of a 10
re carbonyl
19). Illumiical potenright-sidence of retilight onset
hat proteocapable of
physiologe activities
containing
proteorhomain to be

Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the
130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodopsin with archaeal (BR, HR, and SR preﬁxes) and Neurospora crassa (NOP1 preﬁx) rhodopsins (16).
Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin;
BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halobium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp;
Neucra, Neurospora crassa.

!73


!74

Shotgun metagenomics

shotgun
sequence

Metagenomics

!75

articles

Community structure and metabolism
through reconstruction of microbial
genomes from the environment
Gene W. Tyson1, Jarrod Chapman3,4, Philip Hugenholtz1, Eric E. Allen1, Rachna J. Ram1, Paul M. Richardson4, Victor V. Solovyev4,
Edward M. Rubin4, Daniel S. Rokhsar3,4 & Jillian F. Banfield1,2
1

Department of Environmental Science, Policy and Management, 2Department of Earth and Planetary Sciences, and 3Department of Physics, University of California,
Berkeley, California 94720, USA
4
Joint Genome Institute, Walnut Creek, California 94598, USA

RESEARCH ARTICLE

...........................................................................................................................................................................................................................

Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their
roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report
reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other
genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of
genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different
individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level.
The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance
variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous
recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the
pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme
J. Craig Venter,1* Karin Remington,1 John F. Heidelberg,3
environment.
2
2
3

Environmental Genome Shotgun
Sequencing of the Sargasso Sea

Aaron L. Halpern, Doug Rusch, Jonathan A. Eisen,
Dongying Wu,3 Ian Paulsen,3 Karen E. Nelson,3 William Nelson,3
The study of microbial evolution and ecology has been revolutio- fluorescence3in situ hybridization Anthony H. Knap,6 biofilms
Derrick E. Fouts, Samuel Levy,2 (FISH) revealed that all
nized by DNA sequencing and analysis1–3. However, isolates have contained mixtures of bacteria (Leptospirillum, Sulfobacillus and, in
Michael W. Lomas,6 Ken Nealson,5 Owen White,3 and other
been the main source of sequence data, and only a small fraction of a few cases, Acidimicrobium) and1archaea (Ferroplasma 6
Jeremy Peterson,3 Thermoplasmatales). The genome of one
microorganisms have been cultivated4–6. Consequently, focus has members of theJeff Hoffman, Rachel Parsons, of these
shifted towards the analysis of uncultivated microorganisms via archaea, Ferroplasma acidarmanus fer1, isolated fromRogers,4
Holly Baden-Tillson,1 Cynthia Pfannkoch,1 Yu-Hui the Richmond
5
cloning of conserved genes and genome fragments directly from mine, has been sequenced previously (http://www.jgi.doe.gov/JGI_
Hamilton O. Smith1
the environment7–9. To date, only a small fraction of genes have been microbial/html/ferroplasma/ferro_homepage.html).
Slides for UC Davis EVE161 Course biofilm (Fig.Jonathan Eisen Winter 2014 was
recovered from individual environments, limiting the analysis of
A pink Taught by 1a) typical of AMD communities

chlorococcus, tha
photosynthetic bio
Surface water
were collected ab
from three sites o
February 2003. A
lected aboard the S
station S” in May
are indicated on F
S1; sampling prot
one expedition to
was extracted from
genomic libraries w
2 to 6 kb were m
prepared plasmid
both ends to!76
provi

Binning challenge

A
B
C
D
E
F
G

T
U
V
W
X
Y
Z

!77

Binning challenge

A
B
C
D
E
F
G

Best binning method: reference genomes


T
U
V
W
X
Y
Z
!78

Case Study - Human Microbiome Metagenomics

ARTICLES

A human gut microbial gene catalogue
established by metagenomic sequencing
Junjie Qin1*, Ruiqiang Li1*, Jeroen Raes2,3, Manimozhiyan Arumugam2, Kristoffer Solvsten Burgdorf4,
Chaysavanh Manichanh5, Trine Nielsen4, Nicolas Pons6, Florence Levenez6, Takuji Yamada2, Daniel R. Mende2,
Junhua Li1,7, Junming Xu1, Shaochuan Li1, Dongfang Li1,8, Jianjun Cao1, Bo Wang1, Huiqing Liang1, Huisong Zheng1,
Yinlong Xie1,7, Julien Tap6, Patricia Lepage6, Marcelo Bertalan9, Jean-Michel Batto6, Torben Hansen4, Denis Le
Paslier10, Allan Linneberg11, H. Bjørn Nielsen9, Eric Pelletier10, Pierre Renault6, Thomas Sicheritz-Ponten9,
Keith Turner12, Hongmei Zhu1, Chang Yu1, Shengting Li1, Min Jian1, Yan Zhou1, Yingrui Li1, Xiuqing Zhang1,
´
Songgang Li1, Nan Qin1, Huanming Yang1, Jian Wang1, Søren Brunak9, Joel Dore6, Francisco Guarner5,
13
4,14
12
10
Karsten Kristiansen , Oluf Pedersen , Julian Parkhill , Jean Weissenbach , MetaHIT Consortium{, Peer Bork2,
S. Dusko Ehrlich6 & Jun Wang1,13
To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here
we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant
microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set,
,150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent)
microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The
genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire
cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are
also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of
functions present in all individuals and most bacteria, respectively.

!79

ARTICLES

40

PC2
•

•

•

•

30

•

••
•

•

Ulcerative colitis
•

•

•

•

•

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•
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PC1

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Healthy

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20
10

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Cluster (%)

•

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Crohn’s disease
•
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0

P value: 0.031
•

•

•

1

•

Figure 4 | Bacterial species abundance differentiates IBD patients and
healthy individuals. Principal component analysis with health status as
instrumental variables, based on the abundance of 155 species with $1%
genome coverage by the Illumina reads in at least 1 individual of the cohort,
was carried out with 14 healthy individuals and 25 IBD patients (21 ulcerative
colitis and 4 Crohn’s disease) from Spain (Supplementary Table 1). Two first
components (PC1 and PC2) were plotted and represented 7.3% of whole
inertia. Individuals (represented by points) were clustered and centre of
gravity computed for each class; P-value of the link between health status and
species abundance was assessed using a Monte-Carlo test (999 replicates).
Slides for UC Davis EVE161 Course Taught by Jonathan Eisen
Almost all (99.96%) of the phylogenetically assigned Winter 2014
genes belonged

Figure 5 | Cluste
were ranked by t
length and copy n
clusters with the
groups of 100 clu
that contains 86%

were within th
This suggests th
(Supplementar
functions impo
We found tw
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Introduction to Microbial Phylogenomics Course EVE161

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