Rapid Sequencing for Prospective Genomic Epidemiology

On Top of Outbreaks: Rapid Sequencing for
Prospective Genomic Epidemiology
ESCMID Study Group for Epidemiological Markers - ESGEM
Dag Harmsen
University of Münster, Germany
Email: dharmsen@uni-muenster.de
Leap-frogging - Ion Torrent Personal Genome Machine
‘Benchtop’ NGS - PGM Ion Chips
– 314 chip (since January, 2011)
• 1.36 M sensors; 100 nt read length; ~ 10 Mb / run
– 316 chip (since July, 2011)
• 6.3 M sensors; 100 nt / EA 200 nt read length; ~ 100 / 200 Mb / run
– 318 chip (Q4, 2011):
• 11 M sensors; 200 nt read length; ~ 1,000 Mb / run
illumina MiSeq Personal Sequencing System
[WWW: www.iontorrent.com/products-ion-pgm]
PGM Sequencing Progress University Münster
Sequencing Throughput
650
•
June 3, 2011
600
– 314 chips, 100 nt
35.6 Mb
258.8 Mb
594.7 Mb
550
German
Meningococcus
outbreak
2001-2006
•
July 15
– 316 chip, 100 nt
•
September 27, 2011
– 316 chip, 200 nt
MBases sequenced
500
450
Dutch
Klebsiella
OXA-48
outbreak 2011
400
350
300
250
200
150
100
German EHEC
outbreak 2011
50
0
1-Jun3
June
1-Jul15
July
1-Aug
August
Date of Run
1-Sep27, 2011
September
Achievements Prospective Genomic Epidemiology
German EHEC Outbreak 2011
University Münster / Life Technologies
year 2011
week
21
22
23
24
25
26
27
28
29
30
BGI / UKE / Crowd-sourcing
& HPA / Univ. Göttingen /
Illumina / PacBio
Achievements Prospective Genomic Epidemiology
German EHEC Outbreak 2011
University Münster / Life Technologies
year 2011
week
21
22
23
24
25
26
27
28
29
30
BGI / UKE / Crowd-sourcing
& HPA / Univ. Göttingen /
Illumina / PacBio
Achievements Prospective Genomic Epidemiology
German EHEC Outbreak 2011
University Münster / Life Technologies
year 2011
week
21
22
23
24
25
26
27
28
29
30
BGI / UKE / Crowd-sourcing
& HPA / Univ. Göttingen /
Illumina / PacBio
Achievements Prospective Genomic Epidemiology
German EHEC Outbreak 2011
University Münster / Life Technologies
‘Tweetom‘
year 2011
week
21
22
23
24
25
26
27
28
29
30
BGI / UKE / Crowd-sourcing
& HPA / Univ. Göttingen /
Illumina / PacBio
MLST+: Genome-wide Gene by Gene Analysis
/ Ridom SeqSphere+
Vision
• WGS & transcriptomics
•‘plain-language’ reports
Problem
• missing data for Sequence
Type (ST) determination
Jolley & Maiden (2010). BIGSdb. BMC Bioinformatics. 11: 595 [PubMed]
PGM Sequencing Two EHEC Strains
Phylogenetic analyses (by ‘quick
and dirty’ assemblies & BIGSdb
MLST+; n = 1.144 core genome genes;
www.mcgenome.net)
•
strain LB226692 (outbreak 2011)
and strain 01-09591 (2001 German
historic HUS causing isolate)
belong to the HUSEC041 complex
•
both strains are only distantly
related to commonly isolated
EHEC serotypes
Model describing the evolution of
the outbreak strain EHEC
O104:H4 and EAEC 55989 (late
1990 Central African Republic) from a
common EHEC ancestor
Mellmann et al. (2011). PLoS One. 6: e22751 [PubMed]
Genomics for Diagnostics
Dutch Klebsiella OXA-48 Outbreak 2011
• since June, 2011 at the Dutch Maasstad hospital in
Rotterdam (NL)
– 98 patients infected
– 28 patients died
– more than 2000 people were at risk of being infected (as of August, 17th) by:
release of cured patients
transfer of patients to other locations
visitors
hospital employees
[RNW]
Dutch National Institute for Public Health and the Environment (RIVM)
was asked to assist the hospital
Screening Test Dutch K. pneumoniae OXA-48
•
The team developing a molecular screening test was composed of:
– Scientists from Dutch National Institute for Public Health and the Environment (RIVM; NL)
– Scientists from Periodontology, University Münster (Germany)
– Bioinformaticians from Life Technologies (U.S.A.) and the Wellcome Trust Sanger
Institute (UK)
clinical isolate
cultivation
Maasstad & RIVM
DNA extraction;
PGM sequencing
Periodontology, UKM
draft assembly; 36
candidate regions
Life Technologies
comparison with
>200 K. genomes;
reduction to 2
candidate regions
multiplex PCR*
based diagnostic
test assay
Sanger Institute
RIVM
* oxa-48, ctx-m-15, housekeeping gene, homo-polymer rich region
Test assay is used by Dutch hospitals to screen the patients and isolate those with a
positive result.
NATURE News Blog. Genomics Identifies Source of Klebsiella Outbreak. August 16, 2011 [Nature]
GenomeWeb. Münster Team Sequences Klebsiella Outbreak on Ion Torrent PGM. August 16, 2011 [GenomeWeb]
Science. Outbreak detectives embrace the genome era (2011). 333: 1818 [Science]
Genomics for Therapeutics
Neisseria meningitidis Epidemic in Four German Counties (2001-2006)
• strains belong to the
hypervirulent MLST clonal
complex ST-41/44, serogroup
B
• spatial stable over the entire
period of study
• incidence rate: 3.1 / 100,000
• no. of cases: 42
Elias et al. (2010). Emerg. Infect. Dis. 16: 464 [PubMed]
Origin of Strains Characterized Previously by MLST,
MLVA, and Serotyping (German outbreak, 2001-2006)
•
German serogroup B meningococci
outbreak isolates from three
different counties
DE9938 (2004)
– N. meningitidis DE9622
– N. meningitidis DE9686
– N. meningitidis DE9938
•
N. meningitidis MC58
– Control isolate
– Serogroup B, ST-32 cc
– NCBI NC_003112
DE9622 (2003)
DE9686 (2004)
Tettelin et al. (2000). Science 287: 1809 [PubMed]
Elias et al. (2010). Emerg. Infect. Dis. 16: 464 [PubMed]
Genomics for Therapeutics
•
•
•
•
3h MIRA de-novo assembly
5 min MLST+ analysis time / strain
(BIGSdb)
no data edition needed
results:
– susceptibility to penicillin and rifampin
– cases most probably preventable by
vaccination with 4CMenB and
MeNZB (Novartis; EMA registration
anticipated for November 2011)
guidance for appropriate selection of
antibiotics
prevention of further spread of
outbreak by vaccination
* MLST – Multi Locus Sequence Typing
eMLST – extended Multi Locus Sequence Typing
AST – Antigen Sequence Typing
4CMenB – Multicomponent Meningococcal Serogroup B Vaccine
AR – Antibiotic resistance
** not found with ‘hybrid assembly’; recombination event
Category
MLST
eMLST
AST
4CMenB
AR
*
Locus
DE9622
abcZ
10
adk
6
aroE
9
fumC
5
gdh
9
pdhC
6
pgm
9
ST
42
cc
ST-41/44cc
aspA
8
carB
32**
dhpS
11
glnA
3
gpm
7
mtgA
7
pilA
5
pip
4
ppk
3
pykA
9
rpiA
1
serC
4
talA
7
PorA VR1/VR27-2,4
porA partial 39
porA full length
26
FetA
1-5
porB partial 3-1
porB full length42
fhbp
14
FHBP
14
penA
1 (S)
rpoB
18 (S)
MC58
4
10
5
4
5
3
2
74
ST-32cc
1
3
5
8
4
5
3
2
9
6
5
3
3
7,16-2
12
2
1-5
3-24
10
1
1
3 )S)
2 (S)
DE9686
10
6
9
5
9
6
9
42
ST-41/44cc
8
9
11
3
7
7
5
4
3
9
1
4
7
7-2,4
39
26
1-5
3-1
42
14
14
1 (S)
18 (S)
DE9938
10
6
9
5
9
6
9
42
ST-41/44cc
8
9
11
3
7
7
5
4
3
9
1
4
7
7-2,4
39
26
1-5
3-1
42
14
14
1 (S)
18 (S)
Our Vision: From Data …
• epidemiologic surveillance (place,
time & person data) & NGS typing
(MLST+; ‘fourth dimension‘)
• on all organisational levels (local,
regional, national, supranational)
©EARSS: MRSA 2005
• geo-coding / geo-mapping (GIS)
• space-time cluster scan
• trend analysis by modelling
• ‘plain-language’ reports for
physicians & public health officers
rapid on the fly creation and sharing of knowledge!
Conclusions
Rapid NGS for Public Health & Clinical Microbiology
• Prospective genomic epidemiology is technical
feasible
– by introduction of ‘Benchtop-NGS’ machines WGS is becoming
achievable for small- and medium-sized laboratories (‘democratizing
NGS’)
• Genomics for diagnostics
– molecular screening tests & ultra-deep sequencing for pathogen
discovery from human tissues
• Genomics for therapeutics
– e.g., antibiotics, pathogenicity profiling, and/or [reverse] vaccinology
• Genomics for early-warning outbreak detection
– MLST+ typing; GIS and space-time clustering; ‘disease weather-map’
• Biggest bottlenecks
– missing data treatment
– bioinformatics for user-friendly & speedy software solutions (‘Bioinformatics for
Dummies’)
– social (e.g., community agreement on typing schema) & legal (e.g., privacy) issues
Acknowledgment RAPID NGS for Public Health
German EHEC
Outbreak 2011
Dutch Klebsiella OXA48 Outbreak 2011
German Meningococcus
Outbreak 2001-2006
(‚proof of principle‘)
(genomics for diagnostics)
(genomics for therapeutics)
Periodontology
Periodontology
Periodontology
Dag Harmsen
Karola Prior
Rafael Szczepanowski
Dag Harmsen
Karola Prior
Rafael Szczepanowski
Dag Harmsen
Sebastian Jünemann
Karola Prior
Rafael Szczepanowski
Institute for Hygiene
Helge Karch
Alexander Mellmann
Shana Leopold
Martina Bielaszewska
Rita Prager
Simone Guenther
Alain Rico
Craig A. Cummings
Yongmei Ji
Stephen F. McLaughlin
Pius M. Brzoska
Jonathan M. Rothberg
Hajo Grundmann
Leo Schouls
Xander Huiskens
Institute for Hygiene & Microbiology
Ulrich Vogel
Heike Claus
Craig Cummings
Stephen F. McLaughlin
Vrunda Sheth
Anjali Shah
Craig Cummings
Thomas Connor
Nicholas Thompson
Project start: 01.01.2012
Under final negotiations
Associated participants: Life Technologies, OpGen, João André Carriço
Münster Medical Genomics Conference 2012
On Top of Outbreaks: Rapid Sequencing for
Prospective Genomic Epidemiology
ESCMID Study Group for Epidemiological Markers - ESGEM
Dag Harmsen
University of Münster, Germany
Email: dharmsen@uni-muenster.de