Bas E. DutilhBacteriófagos: Aspectos básicos y moleculares. Aplicaciones Biotecnológicas

Buenos Aires, June 29th 2015

Viral metagenomics

Metagenomics

Sample

Filter

Microbesor viruses

Viruses and phages

Metagenomics

Sample

Filter

Microbesor viruses

Who/what is there?

Who/what is there?

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

Biological “dark matter”

“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

F2T1 T2

M

F4T1 T2

M

F1T1 T2

M

F3

Reyes et al. Nature 2010

Pathway presence profiles

Virome readsmapping to viral database

Reyes et al. Nature 2010

1 2 3 4 5 6 7 8 9 10 11 121

10

100

1000

10000

Number of samples contributing reads to contig

Num

ber o

f con

tigs

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

Aver

age

dept

h →

Samples →

1 2 3 4 5 6 7 8 9 10 11 121

10

100

1000

10000

crAssphage97,065 bp

29.3% G+C80 ORFs

CaudoviralesDutilh 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 hitunambiguously aligned

2,906 metagenomes1,193 phages (861)blastn≥95% identity≥75 bp hitunambiguously aligned

Ubiquity/abundance plot

Dutilh et al. Nat. Comm. 2014

??????????????????????????????????????? ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????

Virome readsmapping tocrAssphage

Viral database vs crAssphage

Virome readsmapping 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

12

34

56

78

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