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
Throughput.

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