Introduction to Whole Genome Sequencing and its Applications in Microbial Diagnostics Workshop on Whole Genome Sequencing and Analysis, 2-4 Oct. 2017
Whole genome sequencing is currently revolutionising the way microbial diagnostics is carried out Bacterial identification and typing... REA Plasmid Phage profiles typing Morphology Bacteriocin Serotyping typing IS-typing flj-typing RFLP VTNR PFGE MLEE 16S rrna RAPD MLST AFLP 1920 1940 1960 1970 1980 2000 Evolution of typing methods
Whole Genome Sequencing - One technology takes it all!
Whole Genome Sequencing (WGS) Huge potential for investigating, assessing and managing microbiological safety issues and illnesses Level of resolution much higher than previous methods Rapidly declining costs Several countries are currently moving forward with WGS
40 years of sequencing Fred Sanger develops sequencing by enzymatic synthesis using chain-terminating inhibitors (1st gen. sequencing) The first genome of bacteria, Haemophilus influenza, is sequenced (1.8 Mb) The Human Genome Project is launched, planned to take 15 years GS20 sequencer (454/Roche) using pyrosequencing Pyrosequencing (2nd gen. sequencing) First two Human Genome drafts published Genome Analyzer (Solexa/Illumina) using cyclic reversible terminator sequencing Ion torrent bench top sequencing machines PacBio RS machine capable of singlemolecule sequencing (3rd gen. sequencing) ONT MinION weighing less than 100 g 1977 1990 1995 1996 2001 2005 2006 2010 2011 2015 2017
Tremendous growth in public sequence data GenBank Whole Genome Sequences Source: https://www.ncbi.nlm.nih.gov/genbank/statistics/
Current routine microbial diagnostics 1-2 days 1-2 days 1-2 days 1-several weeks Sample Culturing ID Antibiotic resistance Typing
Sequence based microbial diagnostics 1-2 days 2-3 days Sample Culturing Sam WGS ID Resistance Typing Phylogeny + Much more
From Centers for Disease Control and Prevention (CDC) 2013: Whole genome sequencing is used to detect outbreaks caused by Listeria. The method has allowed scientists to: Detect more clusters of Listeria illnesses Solve more Listeria outbreaks while they are still small Link ill patients to likely food sources Identify new food sources of Listeria, such as caramel apples and ice cream CDC is moving forward using WGS for outbreak investigations of Campylobacter, Shiga toxin-producing E. coli, and Salmonella.
Australian study to evaluate the use of WGS for outbreak detection of L. monocytogenes WGS was compared to conventional typing (binary typing, PCR serotyping, MLST, MLVA, and PFGE) Results were highly concordant (>99%) WGS enabled identification of nested clusters within groups of isolates otherwise indistinguishable National risk-alert system was developed to warn epidemiologists required to act on the data
Conclusion: Whole Genome Sequencing (WGS) is poised to become standard methodology in food safety for the identification and characterisation of foodborne pathogens, including AMR organisms.
During two 6-months periods all Multidrug-resistant methicillinresistant S. aureus, vancomycin-resistant E. faecium, E. coli, and P. aeruginosa were sequenced at University Hospital Muenster, Germany Interval I: 645 isolates, interval II: 550 isolate Analysis of transmission rates during interval I showed that rates within the hospital was low => policy of preemptive isolation cancelled Cancellation did not increase transmission rate during interval II, but led to total savings of Euro 200,000
Bioinformatic platforms Unix
Center for Genomic Epidemiology Aim: To provide the scientific foundation for future internet-based solutions, where a central database will enable simplification of whole genome sequence information and comparison to all other sequenced isolates. To develop algorithms for rapid analyses of whole genome DNA-sequences, tools for analyses and extraction of information from the sequence data and web-interfaces for using the tools in the global scientific and medical community.
Advantages + Platform independent + Requires little computer resources + Can be done everywhere Disadvantages - Requires patience
Unique IP adresses per country Where do the users come from?
Remember the 5 and 3 ends of DNA