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Course on Introduction to microbial whole genome sequencing and analysis Mette Voldby Larsen DTU – Center for Biological Sequence Analysis (CBS) Henrik.

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Presentation on theme: "Course on Introduction to microbial whole genome sequencing and analysis Mette Voldby Larsen DTU – Center for Biological Sequence Analysis (CBS) Henrik."— Presentation transcript:

1 Course on Introduction to microbial whole genome sequencing and analysis
Mette Voldby Larsen DTU – Center for Biological Sequence Analysis (CBS) Henrik Hasman DTU – National Food Institute

2 Presentation Henrik Hasman Ph.D. in molecular microbiology (1999)
Has been working at DTU – National Food Institute since 2000 Main topics are antimicrobial resistance and genetic engineering of microorganisms and practical applications of NGS in clinical microbiology.

3 What do we do Applied research in evolution and spread of pathogenic bacteria with focus on antimicrobial resistance and bacterial typing. Drug development for control of infections Development of bioinformatic solutions for especially clinical microbiology. WHO Collaborating Center and EU Reference Laboratory for antimicrobial resistance (EURL AR). Coordinator of COMPARE (Horizon 2020).

4 Mette Voldby Larsen 2002: Cand. scient. in Biology from University of Copenhagen 2007: PhD in Immunological Bioinformatics from Center for Biological Sequence Analysis (CBS), DTU : Assistant professor at CBS, DTU : Associate professor at CBS, DTU > Primary research fields: Developing methods for whole-genome based prediction of microorganism’s type, phenotype, phylogeny ect. Recently also phages. > Teaching, study leader for Human Life Science Engineering

5 More than 150 employees= one of the largest bioinformatics groups within academia in Europe
Web-services runs a total of more than 1 million jobs per month. The flagship is “SignalP”, which predicts protein localization

6 The course Learning objectives:
Understand the most common NGS technologies and terminology. Learn how to prepare raw data from the sequencer for further bioinformatic analysis. Be able to use tools for In silico detection of plasmid, resistance and virulence genes. Be able to perform global and local WGS analysis to determine clonal relationship of bacteria (SNP, ND, MLST). Cases and discussion of relevant literature. Learn about metagenomics in clinical microbiology.


8 Introduction to NGS Today Welcome
Introduktion to Next Generation Sequencing Illumina præsentation Intro to sequencing, raw data and assembly Lunch (Sandwiches) Journal club Introduction to CGE single isolate, single services Computer work w. single isolates and single services Coffee Wrap-up of computer work

9 Introduction to NGS Tomorrow Welcome back Case - VTEC diagnostics
Coffee Introduction to the SNP/ND concept Computer work w. VTEC Lunch (Sandwiches) Wrap-up of computer work Computer work w. CSIPhylogeny and NDtree Batch upload and the pipeline The map Computer work w. batch upload and the map Sponsored dinner in Lyngby at 18.30

10 Introduktion til NGS Friday Welcome back Wrap-up of computer work
Metagenomics Coffee Case - Urine infections CLCBio presentation Computer work w. MGMapper/your own data Lunch (Sandwiches) Implementing NGS in a clinical laboratory Future perspectives and GMI/COMPARE Course evaluation and goodbye

11 And now to you..? Who are YOU?
Where do you come from (country/institution)? Your daily work? Experience with NGS/WGS? Your motivation for joining the course?

12 Introduction to NGS

13 Next Generation Sequencing
One method to rule them all… Give us our sequence..

14 OCR (Optical Character Recognition)

15 1981 £35000 2006 £2600

16 Ray Kurzweil £ + 2-3£ for App…

17 Workflow today at the clinical laboratory

18 Family Genus Species (Subspecies) Serovar Phagetype Ribotype Resistograms PFGE type MLVA type MLST type DNA Microarray analysis Full genomic DNA sequence Identification It is not sufficient to show that two bacterial isolates are indistinguishable by a certain typing method. A possible epidemiological link also has to be identified! The data describing the relevant epidemiological information is called META DATA. Typing Selecting an appropriate typing method can be depending on initial (less discriminatory) pre-typing. And going directly for the most discriminatory method can sometimes be misleading.

19 Typing methods Phenotypic Serotyping (antibodies)
Phage typing (virus susceptibility) Biotyping (ability to grow in different substrates) Antimicrobial resistance Protein profiles Genotypic DNA fingerprint (RAPD, AFLP, ERIC, MLVA) DNA sequencing (MLST, spa, dru, full genome) 19

20 Workflow with WGS at the clinical laboratory
Didelot et al, 2012.

21 DNA sequencing 21

22 DNA sequencing Applied Biosystems (ABI) Genetic analyser
“First Generation” Sequencing machine (capillary Sanger sequencing) 22

23 23

24 Limitations Limitation
The size of DNA fragments that can be read in this way is about 700 bps...and it takes a long time to rum even a few genes..! Problem Most genomes are enormous (e.g 108 base pair in case of human). So it is impossible to be sequenced directly! This is called Large-Scale Sequencing

25 Solution Solution Break the DNA into small fragments randomly
Sequence the readable fragment directly Assemble the fragment together to reconstruct the original DNA Scaffolder gaps Solving a one-dimensional jigsaw puzzle with millions of pieces(without the box) !

26 NGS output Huge numbers of small fragments ( bp)

27 Second generation sequencing

28 Platforms Loman et al, 2012

29 Platforms Loman et al, 2012

30 Oxford Nanopore (MinION)
Next generation sequencing machines 454 Life Sciences (Roche) First Next Generation Sequencing machine Illumina HiSeq/GAII systems High throughput systems Ion Torrent PGM system Low/medium throughput system Oxford Nanopore (MinION) Single-molecule sequencing Illumina MiSeq system Medium throughput system 30

31 Raw DNA sequences Summary of: What it is? Has it been seen before? How we can fight/treat? What is new/unusual? Client side Google maps like view Reports Outbreaks Rough assembly and compression Fine assembly What is already known? Pathogenicity islands Virulence genes Resistance genes MLST type Identification Server side Gene finding Comparison What is novel? Vaccine targets Virulence genes Resistance genes SNPs

32 Workflow with WGS at the clinical laboratory
4-6 hours Metagenomics Modified from Didelot et al., 2012.

33 Wet-Lab Workflow Library Analysis tools DNA purification DNA barcoding


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