1. Noriko Cassman CMGT Rio de Janeiro, Brasil Nov. 17, 2012
Viral metagenomics in marine environments or, Why Perl is Actually Useful
Noriko Cassman
CMGT
Rio de Janeiro, Brasil
Nov. 17, 2012

2. Ignoring the viruses of your environment? Think again!
10^6 bacteria and 10^7 viruses in each ml of seawater
Metagenomics: linking community genotypes and functional potential to ecology
Bacterial vs. viral metagenomic analysis
Viruses don't do actual metabolism

3. Viral Ecology Plus <1% culturable so we use genomics
Viral marker genes (phoH, psb)
Metagenomes: do we get random samples of all viruses?
Targeted metagenomes: let's look at dsDNA phages
<10% similarity of OMZ viruses to databases so we turn to other biology-inspired methods

4. Enter Evolution: DNA phages affect all trophic levels of an ecosystem
Phages are predators and pool of genes for bacteria
Host cells are required for bacteriophage replication
Most abundant replicating entities in the world
Viruses infect all DNA-based organisms. There are even viruses that infect other viruses. So viruses are important in all levels of an ecosystem.
Breitbart et. al. 2011

5. Two Major Phage Lifecycles within the Host Cell
LYTIC CYCLE
Induction
LYSOGENIC CYCLE

6. Phage lysis releases nutrients and structures bacterial communities
LYTIC CYCLE
Viral infection and lysis of cells releases as much as 40% of dissolved organic material back into the ocean by rough estimates.
By infecting according to which cells are available in an environment, phages naturally target the most abundant member of a bacterial community. This is called Kill the Winner dynamics.
However, the big picture of what viruses and especially phage are doing in the ocean is far from complete. For instance, Kill the Winner only takes into account one-to-one phage/host infectivity.

7. Phage lysogeny provides a “horizontal gene pool” for hosts
Transduction in the environment results in the evolution of bacterial genes residing in phage and prophage genomes
The host cell then expresses the successful prophage genes
During the process of transduction, phages cut and package some of the bacterial genome when they resume a lytic nature. Some phage packaging proteins are notoriously sloppy and we have long since harnessed them for use in the laboratory to transfer bacterial genes via viruses.
Prophage refers to the phage before it resumes a lytic nature.
This close genetic relationship between phages and hosts results in pervasive horizontal gene transfer in the ocean.
LYSOGENIC CYCLE

8. Prophage presence may change a host's phenotype
Lysogenic conversion: when prophage gene expression causes drastic phenotypic change
Vibrio cholerae phage CTXφ
60% of bacterial genomes contain at least one prophage
Within bacterial species, the presence of one or more permanent prophage differentiates between strains
Horizontal gene transfer of metabolically active genes within phages sometimes results in lysogenic conversion.
For example, Vibrio bacteria with the phage encoding the cholera toxin becomes virulent.
Many studies have pinpointed prophage genomes within bacterial genomes.
In studies of E. coli, Salmonella, and Vibrio species, the difference between strains within a species is sometimes due to the presence or absence of a permanent prophage.
It remains to be seen if this is an actual cause of strain and maybe also species differentiation, apart from ordinary vertical hereditary transfer of genetic material.

9. Hunting for Prophages in Bacterial Genomes
Well-studied recombination reactions in phages when inserting into bacterial genome
Catalyzed by phage integrases or recombinases
attB
attP
attL
attR
nucleotide repeats
12/25/2017

10. What can we do with a viral metagenomic dataset?
“Classic” metagenomics tells us about viral ecology: taxonomic potential and functional role
But phages coevolve with their bacterial hosts
Lytic phages
Lysogenic prophages
Permanent prophages
So we can use metagenomes to investigate both ecology and evolution of viruses
Random samples are good!

11. Enter OMZs: Phages tend toward lysogeny in extreme environments
Oxygen Minimum Zones are unique, extreme environments
Normal seawater contains 5 ml/L dissolved O
OMZs contain a permanent anoxic layer with <1ml/L dissolved O
We sampled the Chilean OMZ which is a part of the Eastern Tropical Southern Pacific OMZ.
This figure shows oxygen concentrations at 200 m worldwide in 2009.

12. Larger eukaryotes adapt poorly to hypoxic waters
Bacteria may help establish or maintain the low oxygen conditions found in OMZs
Hypothesis: lysogenic phages increase bacterial fitness in the OMZ
Ulloa et al 2011

13. OMZ viral metagenome collection Dec 2009
Collected 80 L water at anoxic 200 m Niskin rosette
Concentrated samples to 500 ml using tangential flow filtration

14. Prophage and viral metagenome extraction
Incubation with Mitomycin C overnight
0.22 μm filtered
“Prophage metagenome” extracted
Incubated without Mitomycin C overnight
“Viral metagenome” extracted
Thurber et al. 2009
+ Mitomycin C

15. OMZ Datasets 2008 2009 Depth St. 3 St. 5 10m or 55m 90m 200m
Brazil data to come (6 more metagenomes)
OMZ Datasets
2008
2009
Depth
St. 3
St. 5
10m or 55m
Viral
90m
200m
Viral+bact
Viral+prophage+bact
Viral+prophage
White et. al. 2012

16. Homology-based Analysis: viral and prophage comparisons
Homology based approaches
Best bidir BLAST results against Phantome database, ACLAME proteins, Integrase database
Metabolism information
Carbohydrate metabolism (link to anaerobic gut phages)
Homology-based Analysis: viral and prophage comparisons
Metagenomic reads
90% identity contigs (NEWBLER)
95% NR clusters (UCLUST)
Best tBLASTx, BLASTx and psiBLAST match percentages
ACLAME db, Phantome proteins, Phantome contigs, custom Integrase db
Best BLASTx match percentages
SEED nr, RefSeq db (MGRAST, RTMG, STAMP)

17. Significantly different level 2 subsystems (from BLASTx against subsystems on MGRAST at evalue<10-5). Fisher’s exact test was used to find the overrepresented functions in each metagenome (DP Asymptotic continuity correction). The viral metagenome (BLUE) and the prophage metagenome (ORANGE) are different. NOTE: excluded phage capsid and phage capsid protein functions.
Viral and prophage metagenomic analyses with homology-based comparisons are a good start

18. Non-homology based Methods: Viral and Prophage Comparisons
Non-homology based approaches
Nucleotide ratio frequencies (from MetaVir)
Cross assembly info (percentage assembled with bact, assembly of singletons)
k-mer frequencies*
Alpha/beta diversity*
Non-homology based Methods: Viral and Prophage Comparisons
Ecology
Dinucleotide ratio frequencies (METAVIR)
Comparisons with public viromes (METAVIR)
Alpha/beta diversity estimates (PHACCS)
Prophage metagenome is good to collect!
K-mer frequencies (custom Perl scripts)
Contig/cluster frequencies (custom Perl scripts)
OMZ virome overlap (bidirectional BLASTn)
Contig mapping from cross-assemblies (NEWBLER, CRASS)

19. BLAST-based Comparison of Viral and Prophage MG with other viromes (Cluster Tree)

20. BLAST-based Comparison of Viral and Prophage MG with other viromes (MDS plot)

21. A tBLASTx-based comparison between OMZ viromes from the MetaVir pipeline. A tBLASTx is a translated protein query against a translated protein subject. A similarity score was computed for each virome pair by summing the best BLAST hit scores. The scoring matrix was clustered using R and the pvclust package and an MDS plot drawn (all through the MetaVir pipeline).

22. Contig Coverage over depths in the OMZ – conserved gene regions

24. Non-homology based approaches
Nucleotide ratio frequencies (from MetaVir)
Cross assembly info (percentage assembled with bact, assembly of singletons)
k-mer frequencies*
Alpha/beta diversity*
Non-homology based methods: Viral and Prophage and Bacterial Metagenomes
Cross-assembly with bacterial metagenomes (NEWBLER, CRASS)
Prophage insertion site identification (PhiSpy)
Virome and microbiome overlap (bidirectional BLASTn)
CRISPR identification (CRISPR recognition tool)

25. Viral genomes contain genes that may become important for bacterial survival
Reyes et al,
Nature 2010

26. Full picture: linking contribution of phage to microbial community metabolisms

27. Preguntas?