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 LarsenDTU – Center for Biological Sequence Analysis (CBS)Henrik HasmanDTU – National Food Institute
2 Presentation Henrik Hasman Ph.D. in molecular microbiology (1999) Has been working at DTU – National Food Institute since 2000Main topics are antimicrobial resistance and genetic engineering of microorganisms and practical applications of NGS in clinical microbiology.
3 What do we doApplied research in evolution and spread of pathogenic bacteria with focus on antimicrobial resistance and bacterial typing.Drug development for control of infectionsDevelopment 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 Larsen2002: Cand. scient. in Biology from University of Copenhagen2007: 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 SequencingIllumina præsentationIntro to sequencing, raw data and assemblyLunch (Sandwiches)Journal clubIntroduction to CGE single isolate, single servicesComputer work w. single isolates and single servicesCoffeeWrap-up of computer work
9 Introduction to NGS Tomorrow Welcome back Case - VTEC diagnostics CoffeeIntroduction to the SNP/ND conceptComputer work w. VTECLunch (Sandwiches)Wrap-up of computer workComputer work w. CSIPhylogeny and NDtreeBatch upload and the pipelineThe mapComputer work w. batch upload and the mapSponsored dinner in Lyngby at 18.30
10 Introduktion til NGS Friday Welcome back Wrap-up of computer work MetagenomicsCoffeeCase - Urine infectionsCLCBio presentationComputer work w. MGMapper/your own dataLunch (Sandwiches)Implementing NGS in a clinical laboratoryFuture perspectives and GMI/COMPARECourse 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?
18 FamilyGenusSpecies(Subspecies)SerovarPhagetypeRibotypeResistogramsPFGE typeMLVA typeMLST typeDNA Microarray analysisFull genomic DNA sequenceIdentificationIt 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.TypingSelecting 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 resistanceProtein profilesGenotypicDNA 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.
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..!ProblemMost 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 directlyAssemble the fragment together to reconstruct the original DNAScaffolder gapsSolving a one-dimensional jigsaw puzzle with millions of pieces(without the box) !
26 NGS outputHuge numbers of small fragments ( bp)
31 Raw DNA sequencesSummary of:What it is?Has it been seen before?How we can fight/treat?What is new/unusual?Client sideGoogle maps like viewReportsOutbreaksRough assemblyand compressionFine assemblyWhat is already known?Pathogenicity islandsVirulence genesResistance genesMLST typeIdentificationServer sideGene findingComparisonWhat is novel?Vaccine targetsVirulence genesResistance genesSNPs
32 Workflow with WGS at the clinical laboratory 4-6 hoursMetagenomicsModified from Didelot et al., 2012.
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