Introduction to Whole Genome Sequencing and its Applications in Microbial Diagnostics

Transcription

1 Introduction to Whole Genome Sequencing and its Applications in Microbial Diagnostics Workshop on Whole Genome Sequencing and Analysis, Mar. 2018

2 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 Evolution of typing methods

3 Whole Genome Sequencing - One technology takes it all!

4 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

5 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

6 Tremendous growth in public sequence data GenBank Whole Genome Sequences Source:

7 Current routine microbial diagnostics 1-2 days 1-2 days 1-2 days 1-several weeks Sample Culturing ID Antibiotic resistance Typing

8 Sequence based microbial diagnostics 1-2 days 2-3 days Sample Culturing Sam WGS ID Resistance Typing Phylogeny + Much more

9 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 Aim: All Listeria, Campylobacter, Shiga toxin-producing E. coli and Shigella, and Salmonella to be sequenced real time by end of 2018 (90,000 in US).

10 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.

11 WGS and Mycobacterium tuberculosis Routine culture-based full characterisation of M. tuberculosis takes weeks. First-line treatment includes four drugs (rifampicin, isoniazid, ethambutol and pyrazinamide), but resistance is on the rise. Culture-free, PCR-based tests have limitations, especially for detecting resistance for second-line drugs. WGS directly from sample (no culturing) could significantly speed up the time to results. Illumina: M. tuberculosis was detected in all samples from which DNA was successfully extracted (39/40); Antibiotic susceptibility predictions was obtained from 24/39 samples and showed perfect concordance with phenotypic tests. Completed in < 48 hours Oxford Nanopore: Votintseva et al J. Clin. Micro. Although not tested on the same samples, results indicate that ONT may offer the same information in hours

12 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

13 Bioinformatic platforms Unix

14 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.

15 Advantages + Platform independent + Requires little computer resources + Can be done everywhere Disadvantages - Requires patience

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17 Unique IP adresses per country Where do the users come from?

18 Remember the 5 and 3 ends of DNA