Course on Introduction to microbial whole genome sequencing and

analysis

Mette Voldby LarsenDTU – Center for Biological Sequence Analysis (CBS)

Henrik HasmanDTU – National Food Institute

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

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).

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

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

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.

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

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

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

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?

Introduction to NGS

Next Generation SequencingOne

method to rule them

all…

Give us our sequence..

OCR (Optic

al Character R

ecognition)

1981£35000

2006£2600

Ray Kurzweil

100-200£ + 2-3£ for App…

Workflow today at the clinical laboratory

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.

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)

Workflow with WGS at the clinical laboratory

Didelot et al, 2012.

DNA sequencing

21

DNA sequencing

22

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

23

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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

NGS output

Huge numbers of small fragments (35-500 bp)

Second generation sequencing

Loman et al, 2012

Platforms

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

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

Workflow with WGS at the clinical laboratory

Modified from Didelot et al., 2012.

4-6 hours

Metagenomics

Wet-Lab Workflow

Analysis tools

DNA purification DNA barcoding

Library