1.
Applications of Whole Genome Sequencing
to Food Safety – Perspective from a
reference laboratory
Dr Tim Dallman
Gastrointestinal Bacteria Reference Unit
7th December 2015

2.
“PHE are a governmental organisation working
with national and local government, industry and
the NHS to protect and improve the nation’s
health”
- Microbiological Characterisation
- Epidemiological Investigation
- ExpertAdvice

3.
3
All isolates of Salmonella & STEC in England & Wales referred centrally to PHE Colindale,
London
All cases of STEC in England & Wales followed with an ESQ
DAY1 – Isolate receipted and grown overnight in broth
DAY2 – High throughput extraction and submission to central sequencing facility
DAY3 -> DAY6 – Quantification, Normalisation, Library Prep, Sequencing
DAY7 – Automated bioinformatics & Validation
HiSeq 2500 high-throughput
machines

4.
Public Health Questions
• What clone is it? e.g. O157, O26, Typhimurium, Typhi etc
• Have we seen this strain before?
• How pathogenic is this strain to humans?
• How resistant to treatment may this infection be?
4

5.
5
• WGS Laboratory Result
Clone Virulence Fine Type

6.
How do we assess similarity
between genomes?
6 Genomic insight into an ongoing outbreak of Shigella flexneri in men who have sex with men
Strain 1
Strain 2
Ref G T A G
Strain 1 G G A A
Strain 2 A G G G
Ref
Strain 1
Strain 2
0.5
Clone specific reference

7.
7 Presentation title - edit in Header and Footer7
Challenges:
• Many CCs
• Hundreds of strains a week
• Rapid, parallel, hands-off analysis
• Real time cluster detection
SnapperDb
Sample
FASTQs
CC11 – O157db
db
db
db
db
CC17 – O103
CC245 – S. flexneri
CC21 – O26
CC442 – O146
…
30 mins - parallel 2 minutes
https://github.com/PHE-GIDIS/

8.
Typing – SNP
SnapperDb - Outputs
• SNP alignments
• Annotated variants
• Genetic distance between all isolates
8
Sequencing Reads
QC
IDENTIFICATION
CHARACTERISATION
Speciate
Antimicrobial resistance
Virulence factors
TYPING
SNP

9.
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http://benfry.com/zipdecode/

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http://benfry.com/zipdecode/

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http://benfry.com/zipdecode/

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http://benfry.com/zipdecode/

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13 Presentation title - edit in Header and Footer
http://benfry.com/zipdecode/

14.
14 Presentation title - edit in Header and Footer
http://benfry.com/zipdecode/

15.
SNPAddress
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1 2
1
2
3
1
2
3
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5
6
1.1.1
1.2.2
1.2.4
1.2.3
2.3.5
2.3.6

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0.3
0.4
0.5
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0 1 2 3 4 5 6 7 8
ProportionofPais
SNP Distance
Same Person
Household
Epi Linked
N=129, STEC O157:H7 Dallman 2015, CID

17.
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50
100
150
200
250
300
350
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100
Frequencyofpairs
SNP Distance
> 30 days
7 to 30 days
<7 days
Common Source Threshold (CST)
Dallman 2015, CID

18.
Have we identified previously unidentified links?
18
• 160 / 334 (2012) strains fell within 5 SNP CST
• Comprises 53 clusters
– 20 clusters household or same patient
– 33 community clusters
• 20 / 33 clusters were NOT identified through PH investigations carried out of
the time.

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250
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400
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Averagepairwiseresidentialdistance(km)
Cluster Size
WGS Linked Cluster
Epi Linked Cluster

20.
Outbreak – O157:H7
18.35.397.765.1482.X
• 40 cases found within a 5SNP cluster
• 18.35.397.765.1482 – (MODE SNP = 0, MAX SNP = 2)
• stx2a
• AMR fully susceptible (other than tellurite)
20

21.
What’s the neighborhood like?
18.35.397.765.1482
Dallman, Microbial Genomics 2015

22.
0.02
-E1682410--18.35.805.1469.1714.1758.1914-PT8-2c
-143530_H15330045101-1-H153300451-18.35.397.765.1482.2436.2622-PT-8-Thames-Valley
-H123680362--18.35.675.1246.1490.1534.1690-PT1-negative
-143515_H15328036901-1-H153280369-18.35.397.765.1482.2436.2622-PT-8-Greater-Manchester
-143513_H15328036701-1-H153280367-18.35.397.765.1482.2436.2622-PT-8-Yorkshire-and-Humber
-143966_H15332053901-2-H153320539-18.35.397.765.1482.2436.2622-PT-8-Anglia-and-Essex
-134255_H15296039301-1-H152960393-18.35.1103.2050.2337.2396.2564-RDNC-Sussex,-Surrey-and-Kent
-143962_H15332053301-2-H153320533-18.35.397.765.1482.2436.2622-PT-8-Anglia-and-Essex
-H123720471--18.35.380.1387.1631.1675.1831-PT1-negative
-H123700370--18.35.380.913.1157.1200.1355-PT1-negative
-143973_H15332054701-2-H153320547-18.35.397.765.1482.2436.2630-PT-8-Cheshire-and-Merseyside
-143972_H15332054601-2-H153320546-18.35.397.765.1482.2436.2622-PT-8-Cheshire-and-Merseyside
-H124120510--18.35.380.738.982.1024.1660-PT1-negative
-H123660388--18.35.445.1336.1580.1624.1780-PT4-2c
-143529_H15330045001-1-H153300450-18.35.397.765.1482.2436.2622-PT-8-Cheshire-and-Merseyside
-H123920479--18.35.445.842.1086.1129.1284-PT4-2c
-143512_H15328036601-1-H153280366-18.35.397.765.1482.2436.2622-PT-8-Cheshire-and-Merseyside
-H122540610--18.35.397.765.1009.1051.1205-PT1-2a
-136701_H15296039303-2-H152960393-18.35.1103.2050.2337.2396.2564-RDNC-Sussex,-Surrey-and-Kent
-E1686280--18.35.957.1775.2024.2070.2227-PT4-2c
-143965_H15332053801-2-H153320538-18.35.397.765.1482.2436.2622-PT-8-Bedfordshire,-Hertfordshire-and-Northamptonshire
-H124900648--18.35.603.1111.1355.1398.2289-PT1-negative
-143942_H15330045801-2-H153300458-18.35.397.765.1482.2436.2622-PT-8-Wales
-H124440482--18.35.380.738.982.1024.1660-PT1-negative
-H123380760--18.35.713.1310.1554.1598.1754-PT4-2c
22980
-H124640336--18.35.603.1111.1355.1398.1554-PT1-negative
-H124920472--18.35.445.1235.1479.1523.1679-PT4-2c
-WX013999S01E--18.35.275.542.772.808.958-PT4-2c
-121439_H15244047803-1-H152440478-18.35.380.738.2192.2241.2402-PT-1-West-Midlands
-H110141083--18.35.397.765.1482.1612.1768-PT1-2a
-H124160501--18.35.397.765.1009.1051.1211-PT1-2a
-143504_H15326038501-1-H153260385-18.35.397.765.1482.2436.2636-PT-8-Lincolnshire,-Leicestershire,-Nottinghamshire-and-Derbyshire
-H124620303--18.35.972.1802.2053.2099.2256-PT4-2c
-143938_H15330045401-2-H153300454-18.35.397.765.1482.2436.2622-PT-8-Anglia-and-Essex
-H122800318--18.35.397.765.1116.1159.1314-PT1-2a
-143939_H15330045501-2-H153300455-18.35.397.765.1482.2436.2622-PT-8-Anglia-and-Essex
-H122820642--18.35.445.842.2077.2123.2280-PT4-2c
-H124040468--18.35.380.738.982.1024.1178-PT1-negative
-H130260423--18.35.397.765.1482.1526.1682-RDNC-2a
-143940_H15330045601-2-H153300456-18.35.397.765.1482.2436.2622-PT-8-Wales
-143975_H15332055501-2-H153320555-18.35.397.765.1482.2436.2622-PT-8-Anglia-and-Essex
What’s the neighborhood like?
• No travel
• UK Cattle isolates
• 18.35.397.765 has a strong
NW geographical signal
including some possible
environmental exposures
18.35

23.
23

24.
24

25.
In depth interviews & further
investigations
• Revealed 2 supermarket chains and several specific salad products
implicated
• One common ingredient
• Food trace-back information indicates 3 possible growers of this ingredient
including 1 in the region similar strains are enriched in
• Sampling on-going………
25

26.
WGS led surveillance
• Identify linked strains with unprecedented sensitivity and specificity
• Provide context about strains
• Native / Imported
• Geographical signals
• Virulence potential
• Need global databases – clinical, animal, food
NCBI BioProject PRJNA248042
27

27.
28
SURVEILLANCE
RISK ASSESSMENT
INVESTIGATION

28.
Questions to the enteric reference laboratory?
• “Is my isolate related to anything else?”
real-time forensic national surveillance
• So we have an outbreak! Does this new isolate need to
be followed up? Is it in or out!?
rapid frontline confirmation
• How far down the rabbit hole does this thing go!?
cross border collaboration
29

29.
Salmonella Enteritidis 14B
Heartlands Hospital
Birmingham

30.
31
Illumina – sequencing by synthesis
98% Accuracy
100-300 bp read length
1.5 billion reads per flow cell
27 hrs

31.
32
Oxford Nanopore – Nanopore sequencing

32.
Rapid Frontline Conformation
• minION sequencer deployed at hospital with > 30 cases infected
• Using real-time sequence analysis could we identify if the hospital outbreak
was the same strain as the national outbreak?
• 2 samples – 1 Outbreak / 1 Sporadic
• Every 10 mins – extract minION reads and attempt to identify species,
serogroup and strain level resolution
33

33.
Species ID
Align minION reads to taxa
specific genes (MetaPhlA2) using
LAST.
S.enterica identified in under 30
mins
Serogroup ID
Align minION reads to S.enterica
reference genome. Phylogenetic
placement with pplacer
Within 50 mins we could identify
the strains as serotype Enteritidis

34.
Outbreak ID
Alignment of minION reads to
Salmonella Enteritidis reference
genome. Phylogenetic placement
with pplacer
Within 100 minutes the outbreak
strains was unambiguously part of
the national 14b cluster (RED) and the
sporadic cases (BLUE) was indeed
sporadic
Quick et al – Genome Biology 2015

35.
Inns et al – Eurosurveillance 2015

36.
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1.2.3.38.38 – 10 SNP Cluster

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OUTBREAK 4
*
OUTBREAK 1
OUTBREAK 3
OUTBREAK 4
OUTBREAK 2
OUTBREAK 4
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 1
OUTBREAK 3
OUTBREAK 3
OUTBREAK 5
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 1
OUTBREAK 4
OUTBREAK 3
OUTBREAK 2
*
OUTBREAK 2
OUTBREAK 3
OUTBREAK 2
OUTBREAK 4
*
OUTBREAK 3
*
*
OUTBREAK 3
*
OUTBREAK 3
OUTBREAK 3
*
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
OUTBREAK 4
OUTBREAK 3
OUTBREAK 3
OUTBREAK 2
OUTBREAK 2
OUTBREAK 3
*
OUTBREAK 2
OUTBREAK 5
*
OUTBREAK 2
*
*
OUTBREAK 1
OUTBREAK 1
OUTBREAK 3
OUTBREAK 2
*
OUTBREAK 2
OUTBREAK 1
OUTBREAK 3
*
OUTBREAK 2
*
OUTBREAK 2
OUTBREAK 1
*
OUTBREAK 3
*
OUTBREAK 1
*
*
OUTBREAK 1
OUTBREAK 3
OUTBREAK 3
*
*
OUTBREAK 3
OUTBREAK 3
*
OUTBREAK 1
OUTBREAK 3
*
OUTBREAK 4
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 1
*
OUTBREAK 4
*
OUTBREAK 3
*
OUTBREAK 3
*
OUTBREAK 3
*
*
*
OUTBREAK 1
OUTBREAK 2
OUTBREAK 3
OUTBREAK 3
OUTBREAK 2
*
OUTBREAK 4
OUTBREAK 1
*
OUTBREAK 1
*
*
*
OUTBREAK 3
*
*
*
OUTBREAK 2
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
*
*
OUTBREAK 4
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
OUTBREAK 1
OUTBREAK 3
OUTBREAK 1
OUTBREAK 4
*
OUTBREAK 5
OUTBREAK 1
OUTBREAK 3
*
OUTBREAK 4
*
*
*
*
OUTBREAK 1
OUTBREAK 3
OUTBREAK 3
OUTBREAK 4
OUTBREAK 2
OUTBREAK 2
*
*
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
*
OUTBREAK 3
*
OUTBREAK 4
*
*
OUTBREAK 5
OUTBREAK 4
*
*
OUTBREAK 2
*
OUTBREAK 3
*
OUTBREAK 2
*
OUTBREAK 2
OUTBREAK 4
OUTBREAK 4
OUTBREAK 5
*
OUTBREAK 5
OUTBREAK 3
*
OUTBREAK 4
OUTBREAK 1
*
OUTBREAK 4
OUTBREAK 4
OUTBREAK 1
*
*
*
OUTBREAK 5
OUTBREAK 2
*
OUTBREAK 1
*
*
OUTBREAK 3
OUTBREAK 1
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
*
OUTBREAK 3
*
*
OUTBREAK 1
OUTBREAK 1
*
*
OUTBREAK 1
*
*
OUTBREAK 1
OUTBREAK 4
*
*
OUTBREAK 3
OUTBREAK 4
*
*
*
*
OUTBREAK 2
*
*
OUTBREAK 4
OUTBREAK 3
*
*
OUTBREAK 1
OUTBREAK 1
*
*
OUTBREAK 4
OUTBREAK 3
*
OUTBREAK 4
OUTBREAK 3
*
OUTBREAK 3
OUTBREAK 4
OUTBREAK 2
OUTBREAK 1
*
*
OUTBREAK 4
*
*
OUTBREAK 4
OUTBREAK 4
OUTBREAK 4
OUTBREAK 1
OUTBREAK 3
*
OUTBREAK 1
OUTBREAK 1
*
*
*
OUTBREAK 2
*
*
OUTBREAK 3
OUTBREAK 2
*
*
OUTBREAK 3
OUTBREAK 1
OUTBREAK 4
*
*
OUTBREAK 2
OUTBREAK 2
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
OUTBREAK 2
*
*
*
OUTBREAK 1
OUTBREAK 3
*
OUTBREAK 3
*
OUTBREAK 4
OUTBREAK 5
OUTBREAK 1
OUTBREAK 1
*
*
*
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 1
*
*
OUTBREAK 3
*
OUTBREAK 4
OUTBREAK 1
OUTBREAK 2
OUTBREAK 3
*
OUTBREAK 3
OUTBREAK 1
OUTBREAK 3
*
*
*
OUTBREAK 4
OUTBREAK 1
OUTBREAK 1
OUTBREAK 1
OUTBREAK 3
*
OUTBREAK 4
OUTBREAK 4
OUTBREAK 3
OUTBREAK 1
*
*
*
OUTBREAK 3
*
*
*
OUTBREAK 2
*
OUTBREAK 4
OUTBREAK 1
81094_H14372077201-1
OUTBREAK 2
OUTBREAK 2
OUTBREAK 3
*
*
OUTBREAK 1
OUTBREAK 2
*
OUTBREAK 3
OUTBREAK 3
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 2
OUTBREAK 1
*
OUTBREAK 2
OUTBREAK 3
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
OUTBREAK 1
OUTBREAK 4
OUTBREAK 3
*
OUTBREAK 1
OUTBREAK 3
*
*
*
*
OUTBREAK 4
OUTBREAK 3
*
OUTBREAK 1
*
*
OUTBREAK 3
OUTBREAK 4
*
*
OUTBREAK 3
*
*
OUTBREAK 1
*
*
OUTBREAK 1
OUTBREAK 2
OUTBREAK 4
OUTBREAK 4
OUTBREAK 2
OUTBREAK 3
OUTBREAK 1
*
OUTBREAK 3
OUTBREAK 3
OUTBREAK 3
OUTBREAK 2
*
OUTBREAK 1
OUTBREAK 3
OUTBREAK 3
OUTBREAK 2
A
B
C
D
E
X
A
B
C
D
E

39.
Association between phylogeny and
distribution network
• Measure distance between cases on phylogeny and traceback network
• Test association between distance matrices using a non-parametric Mantel
test
• Adjust for cases from same point source outbreak as a covariate
Salmonella NGS at PHE

40.
Can we explain this association?
• Trace back in UK – All outbreaks involve eggs
from same German supplier
• Cases in Austria, Germany, France and
Luxemburg
• Trace back investigations lead to sampling of
egg production premises in Germany
• Company has 4 separate egg production plants
supplied with layer flocks from single supplier
• European isolates sequenced – cases / eggs
41

41.
0.02
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**
*
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*
Clade 1 – 12 egg isolates + traceback (all site A)
Clade 2 – 2 egg isolates + traceback (all site B)
Clade 3 – 0 egg isolates, 0 traceback to site A or B
1
2
3

42.
Hours& Days& Weeks&
Hospital&Regional&Na5onal&Intl’l&
Salmonella

43.
Acknowledgements
44
PHE
Elizabeth de Pinna, Satheesh Nair, Kathie Grant, Phil
Ashton, Claire Jenkins, Martin Day, Neil Perry Cath
Arnold and sequencing team, Jonathan Green,
Anthony Underwood and bioinformatics team, Jeremy
Hawker, Lisa Byrne
Imperial College
Thibaut Jombart
University of Birmingham
Nic Loman, Josh Quick
Roslin Institute
David Gally
Robert Koch Institut
Wolfgang Rabsch, Sandra Simon