course on introduction to microbial whole genome sequencing and analysis mette voldby larsen dtu –...
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Course on Introduction to microbial whole genome sequencing and
analysis
Mette Voldby LarsenDTU – Center for Biological Sequence Analysis (CBS)
Henrik HasmanDTU – National Food Institute
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Presentation
• 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.
• Henrik Hasman
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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).
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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
2007-2012: Assistant professor at CBS, DTU
2012 - : 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
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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
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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.
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Introduction to NGSToday
WelcomeIntroduktion to Next Generation Sequencing
Illumina præsentationIntro to sequencing, raw data and assembly
Lunch (Sandwiches)
Journal club
Introduction to CGE single isolate, single servicesComputer work w. single isolates and single services
CoffeeComputer work w. single isolates and single services
Wrap-up of computer work
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Introduction to NGSTomorrowWelcome back
Case - VTEC diagnosticsCoffee
Introduction to the SNP/ND concept Computer work w. VTEC
Lunch (Sandwiches)
Wrap-up of computer workComputer work w. CSIPhylogeny and NDtree
CoffeeBatch upload and the pipeline
The mapComputer work w. batch upload and the map
Sponsored dinner in Lyngby at 18.30
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Introduktion til NGSFriday
Welcome backWrap-up of computer work
Metagenomics Coffee
Case - Urine infectionsCLCBio presentation
Computer work w. MGMapper/your own data
Lunch (Sandwiches)
Computer work w. MGMapper/your own dataWrap-up of computer work
Implementing NGS in a clinical laboratoryFuture perspectives and GMI/COMPARE
Course evaluation and goodbyeCoffee
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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?
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Introduction to NGS
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Next Generation SequencingOne
method to rule them
all…
Give us our sequence..
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OCR (Optic
al Character R
ecognition)
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1981£35000
2006£2600
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Ray Kurzweil
100-200£ + 2-3£ for App…
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Workflow today at the clinical laboratory
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Family
Genus
Species
(Subspecies)
Serovar
Phagetype
Ribotype
Resistograms
PFGE type
MLVA type
MLST type
DNA Microarray analysis
Full genomic DNA sequence
Identification
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.
It is not suffic
ient to show that tw
o bacterial isolates are
indistinguishable by a certain typing method. A possible
epidemiological link also has to be identifie
d!
The data describing the relevant epidemiological
information is called META DATA.
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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)
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Workflow with WGS at the clinical laboratory
Didelot et al, 2012.
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DNA sequencing
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Applied Biosystems (ABI) Genetic analyser“First Generation” Sequencing machine (capillary Sanger sequencing)
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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
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• 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) !
Solution
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NGS output
Huge numbers of small fragments (35-500 bp)
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Second generation sequencing
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Loman et al, 2012
Platforms
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Loman et al, 2012
Platforms
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Illumina HiSeq/GAII systemsHigh throughput systems
454 Life Sciences (Roche)First Next Generation Sequencing machine
Illumina MiSeq systemMedium throughput system
Ion Torrent PGM systemLow/medium throughput system
Next generation sequencing machines
Oxford Nanopore (MinION)Single-molecule sequencing
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Rough assemblyand compression
Raw DNA sequences
Gene findingComparison
Identification
Fine assembly
What is already known? Pathogenicity islands Virulence genes Resistance genes MLST type
What is already known? Pathogenicity islands Virulence genes Resistance genes MLST type
Google maps like view
• Reports Outbreaks
Summary of:
What it is? Has it been seen before? How we can fight/treat? What is new/unusual?
Serv
er s
ide
Clie
nt s
ide
What is novel? Vaccine targets Virulence genes Resistance genes SNPs
What is novel? Vaccine targets Virulence genes Resistance genes SNPs
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Workflow with WGS at the clinical laboratory
Modified from Didelot et al., 2012.
4-6 hours
Metagenomics
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Wet-Lab Workflow
Analysis tools
DNA purification DNA barcoding
Library
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