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9/15/2008
1
Introduction toIntroduction to
Metagenomics
Phil HugenholtzPhil Hugenholtz
Microbial Ecology Program
History of the “meta” part
Cell genomic cartoon
Carl Woese
9/15/2008
4
extraction
whole–cell hybridization
environmental sample
bulk DNA / RNA
PCR
cloningprobe design
nucleic acid hybridization
community rRNA / rDNA
nucleic acid probes
phylogenetic trees
sequencingcomparative analysis
rRNA / rDNA sequences and database
rRNA / rDNA clones
Bacteria,1998
9/15/2008
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extraction
whole–cell hybridization
environmental sample
bulk DNA / RNA
PCR
cloningprobe design
nucleic acid hybridization
community rRNA / rDNA
nucleic acid probes
phylogenetic trees
sequencingcomparative analysis
rRNA / rDNA sequences and database
rRNA / rDNA clones
genomic
genomic
isolate community
Genomics Metagenomics
sequencing
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The catch…. it comes in small piecesThe catch…. it comes in small pieces
Average Sanger read length - 750 bases
Must assemble the reads together
Giant jigsaw puzzles
Metagenome assembly - like putting together several jigsaw puzzles
Falk Warnecke, JGI
9/15/2008
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. . . with some pieces missing
Falk Warnecke, JGI
Can we still reconstruct?
Falk Warnecke, JGI
9/15/2008
8
Can we still reconstruct?
Falk Warnecke, JGI
Making metagenomes
Sample Extract DNA Shotgun clone High throughput sequence
3, 8, 40 kb
Assemble reads Call genes Bin fragments
9/15/2008
9
Will this really work??
need to test whole genome shot-gun sequencing on a very simple community to see if it will work!
pink biofilm community
Archaea
Sulfobacillus
ArchaeaLeptospirillum
10%4%
1%
Eucarya
85%
FISH countsTyson et al., 2004 Nature 428:37-43
9/15/2008
10
MetabolismExpected
proton pumps for pH
metal resistance genes
novel cytochromes for Fe2+ oxidation2chemotaxis genes for Fe2+ and O2
genes for CO2 and N2 fixation*
lots of amino acid and sugar transporters in the archaea
Unexpected* l t f if*only one set of nif genes
DNA photolyase genes
cellulose synthase in Leptospirillum -biofilm bouyancy
Fine-scale population structure
Shah et al (2005) BMC Bioinformatics 6:29Shah et al., (2005) BMC Bioinformatics 6:29
9/15/2008
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Gross-scale population structure
- align metagenomic reads against reference g g ggenomes or genome fragments
- color-code to enhance visualization
- recently implemented in IMG/M
from Fig. 2 Coleman et al. (2006) Science 311(5768):17
does it work with more complex communities?
9/15/2008
12
Acid mine drainage Sargasso Sea Soil
1 10 100 1000 10000Species complexity
Susannah Tringe
90
100
30
40
50
60
70
80
% Sequence of Reads
0
10
20
Acid Mine DrainageBiofilm
Sargasso Sea Soil
Environmental Sample (Complexity)
(Low)(Moderate) (High)
Gene Tyson
9/15/2008
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Acid mine drainage Sargasso Sea Soil
1 10 100 1000 10000Species complexity
?Susannah Tringe
Environmental Gene Tags (EGT)
Identify genes in sequence data (contigs to unassembled reads) from multipleenvironmental samples
Assign genes to their gene family, or higher level groupings
Compare relative abundance of different gene families according to habitat
9/15/2008
14
A
Adaptive gene for habitat AAdaptive gene for habitat BEssential gene
B
Environmental Gene Tags(EGTs)
Susannah Tringe
proteorhodopsins
hypotheticalsTringe et al. Science Mar 2005
9/15/2008
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Tringe et al. Science Mar 2005
K+ transportNa+ transport
Tringe et al. Science Mar 2005
photosynthesis
antibiotics
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Snapshot studies
xspace
~90% of all current and pending metagenomic
j t
time
projects
Spatial series studiesHypersaline mat
xxxxx
space
xx
time
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Temporal series studies
Ventilator associated pneumonia
x x x x x x xspace
time
New sequencing technologies
Sample Extract DNA Shotgun clone High throughput sequence
pyrosequencing
3, 8, 40 kb
Assemble reads Call genes Bin fragments
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454 pyrosequencer
Sanger GS20 FLX XLR0.07 Mbp 35 Mbp 100 Mbp 400 Mbp per run700 bp 100 bp 200 bp 400 bp reads0.1 ¢ 0.03 ¢ 0.01¢ 0.003 ¢ per base
Mega-metagenomics
600 Mbp alone!
Future?????
9/15/2008
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ProteinsMetaproteomics
Unexplored territory
3 3 3
Enhancing “basic” metagenomics
DNAMetagenomics
RNAMetatranscriptomics
AUG
AUG
AUG
AUG
AUG
AUG
1
2
3
1
2
3
1
2
3
(WGA) WGA
AUG
AUG
AUG
AUG
AUG
AUG
AUG
AUG
AUG
AUG
AUG
AUG
AUG
AUG
SIP
SIP
Cells
Microbialcommunity
Enrichedpopulation
Singlecell
Basic metagenomicapproach
( )
ab