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

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 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 Computer work w. single isolates and single services

Wrap-up of computer work

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

Coffee Batch upload and the pipeline

The map Computer work w. batch upload and the map

Sponsored dinner in Lyngby at 18.30

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)

Computer work w. MGMapper/your own data Wrap-up of computer work

Implementing NGS in a clinical laboratory Future perspectives and GMI/COMPARE

Course evaluation and goodbye Coffee

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 Sequencing One

method to rule them

all…

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.

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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)

Workflow with WGS at the clinical laboratory

Didelot et al, 2012.

DNA sequencing

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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

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 systems High throughput systems

454 Life Sciences (Roche) First Next Generation Sequencing machine

Illumina MiSeq system Medium throughput system

Ion Torrent PGM system Low/medium throughput system

Next generation sequencing machines

Oxford Nanopore (MinION) Single-molecule sequencing

Rough assembly and compression

Raw DNA sequences

Gene finding Comparison

Identification

Fine assembly

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 si

de

Clie

nt si

de

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

Wet-Lab Workflow

Analysis tools

DNA purification DNA barcoding

Library