1. Whole Genome Sequencing for Epidemiologists – A Brief Introduction
Joel R Sevinsky, PhD

2. Objectives Microbial genomes
Common isolate identification techniques using molecular biology
Whole genome sequencing (WGS)
Example of WGS for outbreak investigation
Questions

3. Microbial Genomes Genome size varies from 4.56 to 5.70 Mb
How big is 5 Mb???

4. Harry Potter Story Long story, some big books!
1,084,440 words in all seven books
Average word length ~5 letters
~5,422,200 letters total in box set
E.coli genomes range from 4.56 to 5.70 Mb
A single E. coli 1 box set
Single human 1,000 box sets!!!

5. PFGE (Pulsed Field Gel Electrophoresis)
What do these bands really mean???

6. PFGE (Pulsed Field Gel Electrophoresis)
5 Mb
5Mb
1Mb
0.5Mb
1 site
2 sites
3 sites
4 sites
Restriction enzyme site
Genome size in Mb

7. Harry Potter Story Specific word = enzyme restriction site
Word frequency determines banding pattern.
Different words represent different enzymes.
What does PFGE really tell you then?
Table 1
Book
Frequency
Voldemort (n)
Sorcerer’s Stone 31
Chamber of Secrets 20
Prisoner of Azkaban 37
Table 2
Book
Frequency
Broomstick (n)
Spell (n)
Wand (n)
Wizard (n)
Sorcerer’s Stone 27 14 62 41
Chamber of Secrets 12 6
107 44
Prisoner of Azkaban 20
114 39

8. Isolate Identification Techniques
Protein
Serotyping
PFGE
Pulsed Field Gel Electrophoresis
Total gDNA fragments
16S rRNA
Ribosomal RNA Sequencing
1 gene
MLST
Multi Locus Sequence Typing
7 genes
wgMLST
Whole Genome Multi Locus Sequence Typing
Thousands of reference genes plus pan genome
wgSNP or hqSNP
Whole Genome Single Nucleotide Polymorphism Typing
Total gDNA
DNA
Sequencing
Information 
WGS

9. Whole Genome Sequencing
40 box sets

10. Whole Genome Sequencing
ATGCGTGATCTAGTAGTCTAGGAGCTGACCGATTA

11. WGS for Outbreak Investigations
Salmonella enterica serovar Enteritidis
JEGX01.004
JEGX01.002

12. WGS for Outbreak Investigations
JEGX01.002
JEGX01.004

13. WGS for Outbreak Investigations
A = suspect isolate, same time/PFGE
B = same patient over 5 weeks

14. WGS for Outbreak Investigations
C = suspect isolate for outbreak 5
D = environmental isolate, egg farm swab

15. WGS Beyond Outbreak Investigations
“…comparison of these 61 genomes sequences revealed that neither the 16S gene, nor the gene fragments usually used for MLST, provides biologically meaningful information on the relatedness of the sequenced isolates. The best way to analyze this is by taking into account all the genomic content, rather than looking at one or a few individual genes.”

16. WGS Beyond Outbreak Investigations
Genome size varies from 4.56 to 5.70 Mb
This size variation demonstrates a genomic difference of up to 1 Mb between isolates.
1 Mb = ~1,000 genes

17. WGS Beyond Outbreak Investigations

18. Reference Characterization by WGS
“One Shot” Characterization of STEC
ANI
SerotypeFinder
VirulenceFinder
7-gene MLST
ResFinder
Phylogenetic ID
GENUS/SPECIES: Escherichia coli
SEROTYPE: O104:H4
PATHOTYPE: Shiga toxin producing and Enteroaggregative E. coli (STEC & EAEC)
VIRULENCE PROFILE: stx2a, aggR, aggA, sigA, sepA, pic, aatA, aaiC, aap
SEQUENCE TYPE: ST678
ANTIMICROBIAL RESISTANCE GENES: blaTEM-1, blaCTX-M-15, strAB, sul2, tet(A)A, dfrA7
wgMLST CODE: :

19. Summary of Potential WGS Applications
Outbreak investigation
Sporadic vs outbreak
Not just cluster but phylogenetic relationships
Microbial Source Tracking (MST)
Microbial Surveillance
Food
Environment
Animals, soil, food prep areas, hospitals, etc
Antibiotic resistance monitoring
Genotype predicts phenotype
Mobile vs integrated
Virulence gene monitoring
What else???

20. Questions?