Rapid Sequencing for Prospective Genomic Epidemiology
Transcription
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: [email protected] 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: [email protected]