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Bas E. Dutilh
Bacteriófagos: Aspectos básicos y moleculares. Aplicaciones Biotecnológicas
Buenos Aires, June 29th 2015
Viral metagenomics
Metagenomics
Sample
Filter
Microbes
or viruses
Viruses and phages
Metagenomics
Sample
Filter
Microbes
or viruses
Metagenomics 1.0: database mapping
• General database
• Custom database
– MetaHIT human gut catalogue
– Omnibus of Marine Genes
Taxonomic and functional profiling
Sunagawa et al. Science 2015
Metagenomics 2.0: genomes from metagenomes
• Reference databases fail for most
environmental metagenomes
– “Dark matter”: sequences not in database
• Homology searches fail for many short
sequencing reads
– Fast read alignment tools place upper limit
on evolutionary distance
• Interpretation fails for taxonomic and
functional metagenomic profiles
– Do functions co-occur in a genome?
– To describe interactions between species
you need species/genome-level resolution
• Solution: assembly and binning of (draft)
genomes from metagenomes
“Depending on how they are viewed,
the unknowns can represent either a formidable challenge
or a treasure trove for virus discovery.”
Mokili, Rohwer and Dutilh Curr. Opin. Virology 2012
Unknowns in viral metagenomes
Mokili, Rohwer and Dutilh Curr. Opin. Virology 2012
T1 T2
M
F2
T1 T2
M
F4
T1 T2
M
F1
T1 T2
M
F3
Reyes et al. Nature 2010
Pathway presence profiles
Virome reads
mapping to
viral database
Reyes et al. Nature 2010
1
10
100
1000
10000
1 2 3 4 5 6 7 8 9 10 11 12
Numberofcontigs
Number of samples contributing reads to contig
De novo assembled contigs
6,988 de novo cross-contigs
Family 1 Family 2 Family 3
F1M F1T1 F1T2 F2M F2T1 F2T2 F3M F3T1 F3T2 F4M F4T1 F4T2
220 contigs present in ≥9 samples
Family 4
Averagedepth→
Samples →
1
10
100
1000
10000
1 2 3 4 5 6 7 8 9 101112
crAssphage
97,065 bp
29.3% G+C
80 ORFs
Caudovirales
Dutilh et al. Nat. Comm. 2014
Dutilh et al. Nat. Comm. 2014
%
Genomic position: 0 – 97,065 nt
2,906 metagenomes (940)
blastn
≥95% identity
≥75 bp hit
unambiguously aligned
2,906 metagenomes
1,193 phages (861)
blastn
≥95% identity
≥75 bp hit
unambiguously aligned
Ubiquity/abundance plot
Dutilh et al. Nat. Comm. 2014
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???????????????????????????????????????
???????????????????????????????????????
???????????????????????????????????????
???????????????????????????????????????
Virome reads
mapping to
crAssphage
Viral database vs crAssphage
Virome reads
mapping to
viral database
Dick et al. Genome Biol. 2009
Binning
Paired-end reads
Iverson et al. Science 2012
Binning: Hi-C sequencing
Beitel et al. PeerJ 2014
• Cross-link physically
proximal DNA (formalin)
• Restrict with enzyme
• Ligate restricted sites
• Sequence ligation sites
K-mer binning
Ghai et al. Sci. Rep. 2011
K-mer (k=4) maps (ESOM)
Abe et al. DNA Res. 2005
Depth binning
Speth et al. Frontiers Microbiol 2012
Depth profile binning
Wrighton et al. Science 2012
F1M F1T1 F1T2 F2M F2T1 F2T2 F3M F3T1 F3T2 F4M F4T1 F4T2
Partial nitritation/
anammox reactor
(600 m3)
5.0 m
0.2 m
1.4 m
2.6 m
3.8 m
untreated
washed
granules
1
2
3
4
5
6
7
8
washed
granules
DNA isolation
Organic extraction
Powersoil kit
Organic extraction
Powersoil kit
Organic extraction
Powersoil kit
Organic extraction
Powersoil kit
Sample treatmentSample location DNA isolation
untreated
Differential sampling and DNA extraction
Speth et al. submitted

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