Microbiomes and DNA based studies of microbial diversity - talk by Jonathan Eisen at Singularity University

Microbiomes and DNA based Studies of
DNA based Studies of Microbial Diversity
Microbial Diversity
Jonathan A. Eisen
Jonathan A. Eisen
University of California, Davis
University of California, Davis

1
Friday, March 15, 13

Sequencing and Microbes

• Four major “ERAs” in use of sequencing for
microbial diversity studies
• Each area represented by the Eras is being
revolutionized by new sequencing methods

2

Era I: rRNA Tree of Life

Era I:
rRNA Tree of Life

3

Ernst Haeckel 1866

Plantae
Protista
Animalia

4
www.mblwhoilibrary.org


Whittaker – Five Kingdoms 1969

Monera
Protista
Plantae
Fungi
Animalia
5

Woese

6

Woese

Woese 1987 - rRNA

Microbiological Reviews 51:221
7

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

8

Era II: rRNA in the Environment

Era II:
rRNA in the Environment

9

Great Plate Count Anomaly

10


Culturing Microscopy

11



Count Count
12



Count <<<< Count
13


Solution?


Count <<<< Count
14


Solution?

DNA


Count <<<< Count
15

Analysis of uncultured microbes

Collect from
environment
16

PCR and phylogenetic analysis of rRNA genes

DNA
extraction PCR

Makes lots Sequence
PCR of copies of rRNA genes
the rRNA
genes in
sample

rRNA1
5’
...TACAGTATAGGT
Phylogenetic tree Sequence alignment = Data matrix GGAGCTAGCGATC
GATCGA... 3’
rRNA1 Yeast
rRNA1 A C A C A C
Yeast T A C A G T
E. coli A G A C A G
E. coli Humans Humans T A T A G T

17


DNA
extraction PCR

Makes lots Sequence
the rRNA
genes in
sample

rRNA1
5’
...ACACACATAGGT
Phylogenetic tree Sequence alignment = Data matrix GGAGCTAGCGATC
GATCGA... 3’
rRNA1 rRNA2
rRNA1 A C A C A C
rRNA2 T A C A G T rRNA2
5’
E. coli A G A C A G
...TACAGTATAGGT
E. coli Humans Humans T A T A G T GGAGCTAGCGATC
GATCGA... 3’
Yeast Yeast T A C A G T

18


DNA
extraction PCR

Makes lots Sequence
the rRNA
genes in
sample

rRNA1
5’...ACACACATAGGTGGAGC
TAGCGATCGATCGA... 3’
Phylogenetic tree Sequence alignment = Data matrix
rRNA2
rRNA1 rRNA2
rRNA1 A C A C A C 5’..TACAGTATAGGTGGAGCT
rRNA4 AGCGACGATCGA... 3’
rRNA3 rRNA2 T A C A G T
rRNA3
rRNA3 C A C T G T 5’...ACGGCAAAATAGGTGGA
E. coli Humans rRNA4 C A C A G T TTCTAGCGATATAGA... 3’

Yeast E. coli A G A C A G rRNA4
5’...ACGGCCCGATAGGTGG
Humans T A T A G T
ATTCTAGCGCCATAGA... 3’
Yeast T A C A G T
19


PCR

20

Major phyla of bacteria & archaea (as of 2002)

No cultures

Some cultures
21

The Hidden Majority Richness estimates

Hugenholtz 2002 Bohannan and Hughes 2003
22

Example: Human biogeography

Censored

Censored

23

Era III: Genome Sequencing

Era III:
Genome Sequencing

24

1st Genome Sequence

Fleischmann
et al. 1995 25

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

26

Lateral Gene Transfer

Perna et al. 2003
27

Era IV: Genomes in the environment

Era IV:
Genomes in the Environment

28

l gene own transducer of light stimuli [for example, the kinetics of its photochemical reaction cy-
leDelong Lab
ge- Htr (22, 23)]. Although sequence analysis of cle. The transport rhodopsins (bacteriorho-
iden- proteorhodopsin shows moderate statistical dopsins and halorhodopsins) are character-
roteo- support for a specific relationship with sen- ized by cyclic photochemical reaction se-
from
opsins
ferent.
hereas
philes
r than

rmine
l, we
a coli
pres-
rotein
3A).
nes of
popro-
m was
(Fig.
at 520
band-
erated
odop-
nce of
dth is 29
rption March 15, 13
Friday,

generated

D ownloaded from w
Delong Lab
eorhodop-
resence of
ndwidth is
absorption
. The red-
nm in the
ated Schiff
ably to the

on was de-
s in a cell
ward trans-
in proteor-
nd only in
(Fig. 4A).
edium was
ce of a 10
re carbonyl
19). Illumi-
ical poten-
right-side-
nce of reti-
light onset
hat proteo-
capable of Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the
physiolog- 130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodop-
sin with archaeal (BR, HR, and SR preﬁxes) and Neurospora crassa (NOP1 preﬁx) rhodopsins (16).
e activities Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin;
containing BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halo-
proteorho- bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp;
main to be Neucra, Neurospora crassa.
30
www.sciencemag.org
Friday, March 15, 13 SCIENCE VOL 289 15 SEPTEMBER 2000 1903

of the surface of a single cell3. This in amino-acid sequences were not restricted to the hydrophilic
nt to produce substantial amounts of
efore, the high density of proteorho-
rane indicated by our calculations
otein has a signi®cant role in the MB 0m2
MB 40m5

Monterey Bay and shallow HOT
BAC 40E8
HOT 0m1
MB 20m2
MB 40m12
ed membranes MB 100m10
MB 20m12
BAC 31A8
MB 40m1
MB 100m5
MB 20m5
BAC 64A5
MB 100m7
10 –3 AU

MB 0m1
MB 100m9
10 –1 s
PAL E6
HOT 75m3
e-treated membranes

Antarctica and deep HOT
PAL B1
PAL E7
PAL B2
PAL B8
HOT 75m1
PAL B7
PAL E1
HOT 75m4
PAL B5
stituted membranes PAL B6
HOT 75m8
0.01

Figure 3 Phylogenetic analysis of the inferred amino-acid sequence of cloned
proteorhodopsin genes. Distance analysis of 220 positions was used to calculate the tree
at 500 nm of a Monterey Bay bacterioplankton by neighbour-joining using the PaupSearch program of the Wisconsin Package version
tion of hydroxylamine; middle, after 0.2 M 10.0 (Genetics Computer Group; Madison, Wisconsin). H. salinarum bacteriorhodopsin
C, with 500-nm illumination for 30 min; bottom, was used as an outgroup, and is not shown. Scale bar represents number of substitutions
n in 100 mM phosphate buffer, pH 7.0, followed per site. Bold names indicate the proteorhodopsins that were spectrally characterized in
incubation for 1 h. this study.
32
Friday,
nature.com March 15, 132001 Macmillan Magazines Ltd
© 787

clearly related to Burkholderia (fig. S2) and were found in the main scaffold set, covered
Sargassoof scaffolds representing two dis-
two groups Sea at depths ranging from 4ϫ to 36ϫ (indicated
tinct strains closely related to the published with shading in table S3 with nine depicted in

Fig. 1. MODIS-Aqua satellite image of
ocean chlorophyll in the Sargasso Sea grid
about the BATS site from 22 February
2003. The station locations are overlain
with their respective identiﬁcations. Note
the elevated levels of chlorophyll (green
color shades) around station 3, which are
not present around stations 11 and 13.

Fig. 2. Gene conser- 33
Friday, March 15, 13 closely
vation among

Functional Diversity of Proteorhodopsins?

Venter et al., 34
Friday, March 15, 13 2004

t can be used as an- nomic group using the phylogenetic analysis marker genes, is roughly comparable to the
ish eukaryotic mate- described for rRNA. For example, our data set 97% cutoff traditionally used for rRNA. Thus
nverse relation was
size of the pre-filters
the fraction of se-
table S5). This rela-
sence of 18S rRNA
strong evidence that
deed captured.
es richness. Most
ncultured organisms
ies of rRNA genes
eaction (PCR) with
ed positions in those
small subunit rRNA
ersity of prokaryotic
17). However, PCR-
y biased, because not
th the same “univer-
hotgun sequence data
tified 1164 distinct
es or fragments of
d II assemblies and
orcerer II reads (5).
milarity cutoff to dis-
s, we identified 148
otypes in our sample
he RDP II database
Fig. 6. Phylogenetic diversity of Sargasso Sea sequences using multiple phylogenetic markers. The
y cutoff, this number relative contribution of organisms from different major phylogenetic groups (phylotypes) was
equence similarity is measured using multiple phylogenetic markers that have been used previously in phylogenetic
e predictor of func- studies of prokaryotes: 16S rRNA, RecA, EF-Tu, EF-G, HSP70, and RNA polymerase B (RpoB). The
equence divergence relative proportion of different phylotypes for each sequence (weighted by the depth of coverage
ate with the biologi- of the contigs from which those sequences came) is shown. The phylotype distribution was
efining species (also determined as follows: (i) Sequences in the Sargasso data set corresponding to each of these genes
were identified using HMM and BLAST searches. (ii) Phylogenetic analysis was performed for each
sequence similarity phylogenetic marker identified in the Sargasso data separately compared with all members of that
he accepted standard gene family in all complete genome sequences (only complete genomes were used to control for
icrobes. All sampled the differential sampling of these markers in GenBank). (iii) The phylogenetic affinity of each
to taxonomic groups sequence was assigned based on the classification of the nearest neighbor in the phylogenetic tree.
35

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,
´
Karsten Kristiansen , Oluf Pedersen , Julian Parkhill , Jean Weissenbach , MetaHIT Consortium{, Peer Bork2,
13 4,14 12 10

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.
36
8,16,17
Friday, March 15, 13 that the microbes in our bodies collectively
It has been estimated individuals from the United States or Japan . To get a broader

ARTICLES

40
PC2
•

•
•
30
• ••

Cluster (%)
• • • •

Ulcerative colitis
•
•
•

•
• •
• •

20
• •

•
• •
•

PC1
•

10
• •

•
•
•
•
Healthy
• •

Crohn’s disease
•
P value: 0.031 0
1
•
•
•

• •

Figure 5 | Cluste
were ranked by t
Figure 4 | Bacterial species abundance differentiates IBD patients and
length and copy n
healthy individuals. Principal component analysis with health status as
clusters with the
instrumental variables, based on the abundance of 155 species with $1%
groups of 100 clu
genome coverage by the Illumina reads in at least 1 individual of the cohort,
that contains 86%
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 were within th
inertia. Individuals (represented by points) were clustered and centre of This suggests th
gravity computed for each class; P-value of the link between health status and (Supplementary
species abundance was assessed using a Monte-Carlo test (999 replicates). functions impo
We found tw
Almost all (99.96%) of the phylogenetically assigned genes belonged required in37 b
all
Friday, March 15, 13 to the Bacteria and Archaea, reflecting their predominance in the gut. for the gut. Am

Woese Tree of Life

??????

adapted from Baldauf, et al., in Assembling the
38
Friday, March 15, 13 Tree of Life, 2004

PD: All

From Wu et al. 2009 Nature 462, 1056-1060 39

Microbiomes and DNA based studies of microbial diversity - talk by Jonathan Eisen at Singularity University

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