1. Rob Edwards San Diego State University
Population Genomics
Rob Edwards
San Diego State University
Image: Lisa Brown for National Public Radio

2. Phages in the Worlds Oceans
GOM
41 samples
13 sites
5 years
SAR
1 sample
1 site
1 year
BBC
85 samples
38 sites
8 years
ARC
56 samples
16 sites LI
4 sites

3. Most Marine Phage Sequences are Novel

4. Marine Single-Stranded DNA Viruses
6% of SAR sequences ssDNA phage (Chlamydia-like Microviridae)
40% viral particles in SAR are ssDNA phage
Several full-genome sequences were recovered via de novo assembly of these fragments
Confirmed by PCR and sequencing

5. SAR metagenome and Chlamydia φ4
Individual sequence reads
Coverage
Concatenated
hits
Chlamydia phi 4
genome
Chl4 ORF calls
12,297 sequence fragments hit
using TBLASTX over a ~4.5 kb genome

6. The phage proteomic tree

7. Signature sequences HECTOR and PARIS
Degenerate primers that amplify T7 DNA polymerase
Tested samples from around world
Tested by different investigators in different laboratories
Mya Breitbart

8. T7 phages are globally distributed
~1026 copies of each sequence on the planet
= 60 metric tons of this DNA sequence
Breitbart et al, FEMS Micro Lett

9. Some phages are everywhere
ssDNA
λ-like
Phage
Proteomic
Tree v. 5
(Edwards, Rohwer)
T4-like
T7-like

10. Compare viruses to all metagenomes
Phage P4 – 11kb, 10 ORFs
Azul – individual sequence reads in a metagenome
Verde – coverage across genome

11. Parts of viruses are everywhere
# metagenome
hits
P4 phage genome

12. Viruses have lots of unknown genes
Microbial
Viral

13. Bas Dutilh

14. cross Assembly
metagenome 1
metagenome 2
metagenome 3
metagenome 4

15. cross Assembly metagenome 1 metagenome 2 metagenome 3 metagenome 4

17. HMP viruses
Reyes et al. Nature 2010

18. Phages are more variable than microbes
Reyes et al. Nature 2010
Functions present in samples

21. Complete crAssphage genome

23. Complete crAssphage genome

25. https://twitter.com/Venustwofacecat
What are chimeras?
Chimerization is more frequent between closely related strains
Similar sequences
Venus the chimeric cat
What are intra-phyla chimeras???
Aziz et al. NAR 2010

29. What is the host? Requires correct host strain
Requires phage makes visible plaques
Often requires correct concentrations of Mg++, Ca++, etc
No PCR hits in at least 100 plaques isolated from 10 pooled viral preparations on Bacteroides fragilis and B. thetaiotaomicron lawns.

31. crAssphage is abundant!
Abundance-ubiquity plot

34. Potential caveats Phage or contamination? Amplification skews?
Highly abundant in viral metagenomes size- and density-filtered for VLPs
ORFs show similarity to bacteriophage and bacterial proteins (no conserved bacterial or archaeal metabolic genes)
Phage-like modularity among functions
Coding structure of the ORFs is typical of a phage genome
Putative prokaryotic promoter patterns
Genome detected in many metagenomes around the world
Amplification skews?

35. Summary crAssphage is everywhere everyone has it (rounding up)
we don't know what it does

36. metagenomics metagenomics 1.0: profiling
metagenomics 2.0: population genomics

37. Tools for population genomics
AbundanceBin
CompostBin
CONCOCT
crAss
GroopM
Metabat
mmgenome

38. Discussion points How many genomes would you expect in a population?
More coverage versus more samples?
Cutoffs for inclusion (e.g. GC, closeness, etc)

39. Discussion points
How do you know the contigs are from the same organism (genotype)
BLAST hits
GC content or k-mer composition
Single copy genes
abundance profiles across metagenomes
Paired ends/mate pairs
PCR
PFGE and size comparisons
SIP and metabolically active fraction
Single cell genomics
Culturing / genome sequencing