Use of next-generation sequencing to investigate foot-and ... · Donald King...
19
Donald King [email protected] OIE Reference Laboratories for FMD and SVD Use of next-generation sequencing to investigate foot-and-mouth disease virus transmission
Transcript of Use of next-generation sequencing to investigate foot-and ... · Donald King...
Donald King [email protected]
OIE Reference Laboratories for FMD and SVD
Use of next-generation sequencing to investigate
foot-and-mouth disease virus transmission
Reference Laboratories at the Pirbright Institute
New high-containment laboratory will open in 2014/15
Foot-and-Mouth Disease Virus
• Family Picornaviridae, genus Aphthovirus • Causes a highly contagious disease of cloven-hoofed
livestock Cattle, sheep, goats and pigs
• + sense ss RNA genome ~8300nt • 7 Serotypes (O; A; C; SAT1; 2; 3 and Asia-1)
including numerous subtypes VP1 sequence data widely used for strain characterisation
L 2A 3CPrimary cleavages
Poly(C)
L 1A 1B 1C 1D 2A 2B 2C 3A 3B 3C 3D
Secondary cleavages 1B/RNA? 3C 3C 3C 3C 3C 3C
Kilobases
0 1 2 3 4 5 6 7 8
ProteaseCarboxy-terminalself-cleaving
ProteaseCapsid NTP binding* Polymerase
Genome-linked(VPg)
Membrane-binding
VP4 VP2 VP3 VP1VPG
5’UTR
AAA (n)
3’UTR
• Multiple virus serotypes/topotypes/strains
• Monitoring global patterns of virus distribution Tracing sources of outbreaks
Early recognition of the emergence of new lineages
• Antigenic prediction and vaccine selection
Why do we sequence FMDV?.........
Example: Molecular Epidemiology O/SKR/1/2011
O/SKR/3/2011
O/Paju/SKR/2010 (NVRQS)
O/SKR/13/2010
O/SKR/7/2010
O/SKR/10/2010
O/SKR/12/2010 (KC438373)
O/Yeoncheon/SKR/2010 (NVRQS)
O/Yangju/SKR/2010 (NVRQS)
O/SKR/8/2011
O/CHA/31/2010* (JF792356)
O/JPN/MZ1/2010 (AB618503)
O/HKN/14/2010
O/HKN/7/2010 (JQ070303)
O/HKN/8/2010
O/HKN/13/2010
O/HKN/15/2010
O/HKN/9/2010 (JQ070304)
O/HKN/10/2010
O/HKN/11/2010
O/HKN/12/2010
O/33-P/CHA/2010 (JQ973889)
O/SKR/4/2010 (JQ070320)
O/KOR/1/2010* (HM143846)
O/GZ/CHA/2010 (JN998086)
O/VN/LC169/2009 (HM055510)
O/MY/CHA/2010 (HQ652079)
O/NC/CHA/2010 (HQ652080)
O/GSLX/CHA/2010 (JQ900581)
O/VN/YB10/2010 (HQ260720)
O/VIT/NCVD-8/2010 (NVRQS)
O/VN/YB08/2010 (HQ260718)
O/VIT/NCVD-9/2010 (NVRQS)
O/VN/YB09/2010 (HQ260719)
O/TAI/22/2009 (HQ116269)
O/VIT/NCVD-19/2010 (NVRQS)
O/VIT/NCVD-20/2010 (NVRQS)
O/GZ-MT/CHA/2013 (KJ646655)
O/Primorskiy/RUS/2014 (ARRIAH)
O/SKR/6/2014
O/SKR/01/2014* (APQA)
O/MYA/7/98 (DQ164925)
Mya-98
O/TAI/189/87* (TRRL)
Cam-94
SEA
WA
EA-1
EA-4
EA-3
EA-2
ME-SA
EURO-SA
ISA-2
ISA-1
CATHAY
100100
100
100
99
100
99
8979
77
83
86
100
98
99
96
98
86
97
100
85
82
75
74
89
85
86
80
84
0.02
• July 2014: FMD cases in Rep. of Korea
• FMD virus closely related to recent outbreaks in the Eastern part of the Russian Federation
• Appears to be a separate introduction into South Korea from cases in 2010-11
Limitations of VP1 data
• VP1 sequences can be used to provide evidence to support transboundary movements of FMDV
• Useful for regional and country-level epidemiology
• VP1 not typically useful to resolve transmission trees within outbreak clusters
• Can we use full genome sequence data to increase the resolution of analysis and trace the spread of FMDV during an outbreak?
• Sequencing improvements make rapid full-genome sequencing achievable Sanger methods NGS approaches
Complete genome sequencing work:
Caroline Wright
Guido König
Begoña Valdazo- González
Dan Haydon
Eleanor Cottam
Nick Knowles
Marco Morelli
Richard Orton
Grace Logan
Graham Freimanis
David King
Kasia Bankowska
Antonello Di-nardo
Müge Fırat-Saraç
Faizah Hamid
Jemma Wadsworth
Ack
no
wle
dge
me
nts
:
Cottam et al., 2006, J. Virol
Increased number of informative sites Retrospective analysis of the 2001 outbreak in the UK
Genome position
2A
2B 2C 3A 3B 3C 3D VP4 VP2 VP3 VP1 AAA (n) L
0 8000
• Analysis of 23 complete genome sequences (consensus) • 197 sites with nt substitutions
IAH2
IAH1
AY593815
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP6b
IP1b(1)
IP1b(2)
IP7
MAH IP8
Farm-to-farm level resolution
• Discriminates between viruses recovered from individual farms Data was provided rapidly (in real-time) to support UK eradication
programme Provided evidence for the existence of farms with undisclosed
infection
• Full genome sequences can also resolve transmission events (direct and indirect contact) in individual animals
Cottam et al 2008 PLoS Pathogens
TCS representation of sequences recovered from farms (UK 2007)
Sequenced virus Un-sampled intermediate
Juleff et al., (2013) J. Gen. Virol.
• High error rate of the RNA polymerase
• Viruses exist as a mixture of closely related sequences within cells
• Sequencing methods can dissect population diversity within samples
• Results will improve our understanding of the mechanisms that drive the evolution of FMDV
Beyond the consensus:
Population diversityX X X
X XXXX
XXXX X
XX X
X XXX
Consensus
Sequence
At depth “beyond the consensus”:
• To dissect the viral mixtures “quasispecies” present in a sample
• Evolutionary processes of RNA viruses at the finest scale
At the consensus level:
• Generate lots of viral sequences in a relatively short time
• Exploit multiplex format (unique indexes)
• Pathogen discovery: recognition of unique signatures association with uncharacterised (or unexpected) pathogens
• RNAseq – mRNA transcript analysis
Potential advantages and uses of NGS
Only the best quality reads (50nt) used • 7,190,884 ⋙ 2,828,554 A (run 1) • 10,116,147 ⋙ 8,969,902 B (run 2)
Raw data
Run 1
Run 2
Data used
Wright et al 2011 J. Virol.
Pilot expt: coverage generated from NGS:
Samples: 1. Inoculum 2. Back right foot lesion (BRF) 3. Front left foot lesion (FLF)
KEY: AGCT
Evolution of a cell culture adapted virus Codon changes in cattle:
VP356 VP360
Glycine to Aspartic Acid
GAC
Arginine to Cysteine TGC
Arginine to
Histidine CAC
78.1
21.9
93.2
6.8
100 100 100
66.5
33.5
% N
ucl
eoti
de
(FLF
)
89.7
10.3
85.6
14.4
100 100
74.1
25.9
100
% N
ucl
eoti
de
(BR
F)
100 100 100 100 100 100
% N
ucl
eoti
de
(In
oc)
Arginine Glycine
Wright et. al. 2011 J. Virol.
INNOCULUM Heparan Sulphate
FLF FOOT LESION
Integrin
BRF FOOT LESION
integrin
Patterns of sub-consensus changes: C3 C1 C2
Transmission direction
C4
Lost
Drifting
Fixed
Morelli et al, (2013) Vet. Res.
Global Phylogeography
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!( !(
!(
!(
!(
!(
!(!(
!(!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(!(
!(!(
!(
!(
!( !(
!(!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!( !(!(!(
!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(!( !(
!(
!(
!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(!( !(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(!(
!(!(
!(
!(
!(
!(
!(
!(!(!( !(
!(
!(
!(!(!(
!(!(
!(
!(
!(
!(
!(!(
!( !(
!(
!(!(
!(
!(
!(!(
!(
!(!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!( !(!(
!(
!(
!(
!(
!( !(
!(
!( !(!(
!(
!(
!(
!(!(!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(!(
!(
!(!(
!(
!(
!(!(!(!(!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!( !(!(
!(!(!(
!(
!(!(
!(!(
!(
!(!(
!(!(
!(!(
!(
!(
!( !(!(
!(!(
!(!(
!(!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!( !( !(!(
!(
!(
!( !(
!(
!(
!(!(!( !( !(
!(
!(
!(
!(
!(!(
!(
!(
!(
!( !(
!(!(
!(
!(
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(!(
!(
!(!(
!(
!(
!( !(!(
!(
!(!(
!(!(
!(
!(
!(
!(!( !(!(
!(
!(!(!(!(!(!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(!(
!(
!(
!( !(
!(
!(!(
!(!(
!(
!(
!(!(
!(
!(!(
!(!(
!(!(
!(
!(
!(!( !(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(
!(!(
!(
!(
!(
!(
!(!(!(
!( !(
!(
!(!(!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(!(
!( !(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(!( !(
!(
!(!(
!(
!( !(
!(
!( !(!(!(
!(
!(
!(
!( !(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!( !(!( !(
!(
!(!(!(
!(
!(
!(
!(
!(
!(!(
!(!(
!(!(
!( !(
!(
!(
!(
!( !(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!( !(!(!(
!(
!(
!(
!( !(
!(
!(
!(
!(
!(
!(
!(!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!( !(
!(
!(!(
!(
!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(!(!(
!(
!(
!(!(
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(!(
!(!(
!(!( !(
!(
!(!( !(
!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(
!(
!(
!(!(
!(!(
!(!( !(
!(
!(
!(!(!(
!(
!(
!(
!(!(
!( !(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!( !(!(!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!( !(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(
!( !(
!(!(
!(
!(
!(!(
!(
!(!(!(!(!(!(
!(
!(
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(!(!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(
!(!(
!(
!(
!(!(!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(!(
!(
!(!( !(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!( !(
!(!( !(!(
!(
!( !(!(
!(
!(
!(!(!(
!(!(
!(
!(!(
!(
!(!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(
!(!(
!(
!(
!(
!(
!(
!( !(
!(
!(
!(!(
!(
!(
!( !(!( !(!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(
!(!(!(
!(
!(
!(
!(
!(
!(!(
!(!(!(
!( !(
!(
!(
!(!(
!(!( !(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!( !(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(!( !(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!( !(!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(!(!( !(
!(!(
!(
!(
!(
!(
!(
!( !(
!(
!(!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(!(
!(!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!( !(!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(!( !(
!(
!(
!(!(!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(!( !(
!(
!(
!(!(
!(
!(
!(
!(!(!(!( !(
!(
!(
!(
!(
!(
!( !(
!(
!(!(!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(!(!(!(!(!( !(
!(
!(!(
!(
!(!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(
!(
!(!(
!(!(
!(
!(
!(!(!(!(!(
!(
!(
!(
!(
!(
!(
!( !(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(
!(
!(
!(
!(!(
!(
!(
!(
!(
!(!(
!(
!(
!(!(
!( !(!(
!(
!(
!(
!(
!(
!(
!(!(!(!(
!(!(
!(!(
!(
!(
!(!(
!(!(
!(
!(
!( !(!(!( !(
!(
!(!(!(
!(!(!(
!(
!(!(
!(
!(!(
!(!(
!( !(
!(
!(!(
!(
!(
!(
!(
!(!(!(
!(
!(!(!( !(!(
!( !(
!(!(!(!( !(
!(!(
!(!( !(!(
!(!(!(
!(
!(!(
!(
!(
!(
!(!(!(
!(
!(!(
!( !(!(
!(!(
!(
!(
!(!(
!(!(
!(
!( !(!(
!(
!(
!(
!(
!(!(
!(
!(!(
!(
!(
!(
!(!(
!(!(
!(
!(
!(
!(
!(
!(!(!(!(
!(
!(!( !(
!(!(!(
!(
!(
!(
!(
!(!(
!(
!(
!( !(
!(!(
!(
!(
!(!(!(!( !(
!( !(
!(!(!(!(!( !(!(
!(
!( !( !(!( !(
!(
!(
!(
!(!( !(!( !(
!(!(
!(!(!(!(!(
!(
!(!(!(
!(!(!( !(
!(
!(!(!(
!(!( !(!(!( !(!(
!(
!(!(!( !(!( !(!(!(!( !(!(!(
!(
!(
!(
!(!(
!(!(
!(!(
!(
!(!(!(
!(
!(!(!(
!(!(
!(!(!(!(!(
!(!(!(
!(
!(!( !(!( !(
!(!(
!(
!(
!( !(!(
!(
!(!(
!(
!(
!(
!(!(!(
!(!(
!(
!(!(!(!(!(
!(
!(
!(
!(
!(
intra-cellular dynamics
Population diversity
animal-animal transmission
farm-to-farm spread
Outbreak epidemiology
cell-to-cell infection
within host pathways
X X XX XX
XXXX
XX XX
X XX X
XX
5’ AAAA
•Sequence data for FMDV (VP1 ► complete genome ► deep-seq)
• These different scales are connected
• Underpinned by the error rate of the viral polymerase
• Field sampling, in-vivo and in-vitro experiments can only ever provide an incomplete picture of these processes
Joining the dots and scales
Orton et al., (2013) Phil Trans B
EpiSeq project
Epi-Seq aims to exploit NGS technologies to: • Generate improved tools for use in real-time monitoring of
epidemics Results will bring novel insights into:
Epidemiology: monitor trans-boundary movements Evolutionary ecology: genetic determinants underpinning phenotypes
• Target important RNA/DNA viruses:
Causing epidemic disease (FMDV/AIV) Causing endemic disease (CSFV)
2 DNA viruses (ASFV and Poxviruses)
• Partners: Belgium, Germany, UK, Italy and Sweden and Denmark
NGS (Illuminia) protocol for FMDV
Co
vera
ge/s
ite
Logan et al, (2014) BMC Genomics
• PCR-free protocol o Eliminates requirements for extensive primer panels
• Can be applied to “any” RNA virus with a poly (A) tail • High coverage suitable for consensus and deep-sequencing • Multiplexing (up to 96 samples/run) is possible
• Future advances will deliver new platforms and increased capacity to generate sequence data
• Requires close relationships between molecular virologists, bioinformaticians and informaticians
Goals:
• Improved pipelines to reduce process-error and/or development of models to accommodate error in our data
• Approaches to translate genetic relationships into transmission trees (also using epidemiological data)
• Reliable (statistical) measures of the likelihood of transmission links
Summary and future priorities
• The FMD Reference Laboratory
• Nick Juleff
• David Paton
• Jan Kim
• John Hammond
• Partners on EpiSeq project
Acknowledgements: Work supported by:
Photo courtesy of HDR Architecture, Inc.; © 2104 James Brittain
