S E O S S N S E 4 G U A 1 A 0 I Q M S D I 2 L I E G E E T R N B U D O D O O F T S E C E I X S O R N U I R 7 A A IO T I E L D ES T V U R C A U C E M M R O F O L T N O IN R M ES CT AU Antoine Huguenin D E & Laboratoire de
P S GS N
Parasitologie Mycologie
Bref historique -2000: Premires description cliniques de myctomes (Atharvaveda) 1829: Champignon impliqu dans le favus (Schnlein) 1876: R.Koch, bactrie responsible de lanthrax 1881: R. Koch premier milieu de culture 1884: Coloration de GRAM 1977: Sanger squenage 1986-1988: premiers squenages de lADN 16S 1987 Premier automate de squenage 1988: Tanaka dcrit le principe du MALDI-TOF
Lessors des nouvelles techniques
Depuis 2005: Spectromtrie de masse: PCR/ESI-TOF MALDI-TOF Gnomique Explosion du squenage: NGS (Next-Generation Sequencing) NNGS (Next-Next Generation Sequencing) PCR multiplex/puces ADN.
Pourquoi une telle acceleration?
Pourquoi une telle acceleration?
Hayden ECE. Biodefence since 9/11: The price of protection. Nature.
2011 Sep 7;:1502.
Le Saint-Graal de lidentification En cas dattaque bioterroriste: Identification RAPIDE (<24h) => pas dtape de culture Dtection SIMULTANEE de plusieurs pathognes bactriens, viraux ou fongiques Systme didentification TRANSPORTABLE Detection des facteurs de virulence/rsistances: SOUCHES LETHALISES Dtection de NOUVELLES SOUCHES 6
ECKER D, DRADER J, GUTIERREZ J, GUTIERREZ A, HANNIS J, SCHINK A, et al. The Ibis
T5000 Universal Biosensor: An Automated Platform for Pathogen Identification and Strain Typing. SAGE Publications; 2006 Dec;11(6):34151.
2006: IBIS T5000 Biosensor
2006: IBIS T5000 Biosensor
PLEX-ID (2009)
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PLEX-ID (2009)
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PLEX-ID (2009) Broad Fungal Kit
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MALDI-TOF vs ESI
13 Lavigne J-P, Espinal P, Dunyach-Remy C, Messad N, Pantel A, Sotto A. Mass spectrometry: a revolution in clinical microbiology? Clinical Chemistry and
MALDI-TOF en myco levures
Score-oriented dendrogram of ATCC and DSMZ type strains of Candida species and yeastlike fungi (n = 18) Microflex LT spectrometer.
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Marklein G et al. J. Clin. Microbiol. 2009;47:2912-2917
MALDI-TOF en myco champignons
filamenteux
15 Ranque S, Normand A-C, Cassagne C, Murat J-B, Bourgeois N, Dalle F, et al. MALDI-TOF mass spectrometry identification of filamentous fungi in the clinical laboratory. Mycoses.
MALDI TOF les piges
16 Normand A-C, Cassagne C, Ranque S, L'Ollivier C, Fourquet P, Roesems S, et al. Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi. BMC Microbiol. BioMed Central Ltd; 2012 Dec
MALDI TOF importance de la
librairie
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MS dans un laboratoire de microbiologie
18 Fournier P-E, Drancourt M, Colson P, Rolain J-M, La Scola B, Raoult D. Modern clinical microbiology: new challenges and solutions. Nature Microbiology Review; 2013 Aug
Next generation sequencing (NGS)
Next generation sequencing: plus vite, moins chers, tude du gnome ou du transcriptome complet
Illumina : Genome analyzer/MySeq
Ion Torrent: Personal Genome Machine/Proton Roche 454 sequencing: GS Junior/FLX SOLID Applied biosystem Life technology
NGS Illumina Prparation de la bibliothque: ADN simple brin, avec adaptateurs de ligation
Fixation sur la surface chambre de raction
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NGS Illumina Ligation et synthse du brin complmentaire
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NGS Illumina Dnaturation des fragments doubles brins
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NGS Illumina La librairie est prte !
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NGS Illuma
Squenage : amplified single molecule sequencing
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NGS Ion Torrent
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NGS Ion Torrent
Appareil photo numrique
Millions de pixels, convertissant la lumire en information digitale
Ion Torrent sequencing chip
Millions de puits convertissant un signal
chimique en information digitale
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NGS Ion Torrent
Quand un nuclotide est incorpore
une molcule dADN il y a libration dun proton, responsable dun changement de pH
Les nuclotides sont ajouts
squentiellement Le pH est mesur par le semiconducteur et il est converti en ajout ou non de nuclotide
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NGS Ion Torrent
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NGS Ion Torrent
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NGS Ion Torrent
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NGS Ion Torrent
Copy DNA
Load chip
A sample of DNA is cut
into millions of fragments, and each fragment is attached to its own bead
The beads are then
flowed across the chip, each being deposited into a well
The fragment is copied
until it covers the bead This automated process produces millions of beads covered with millions of different fragments
Then the chip is
flooded with one of the four nucleotides
Incorporate nucleotide
If the next base on the
DNA strand is complementary to this nucleotide, a nucleotide will be incorporated and a hydrogen ion will be released The hydrogen ion changes the pH of the solution in the well
Detect and call
An ion-sensitive layer beneath the well measures that pH change and converts it to voltage This voltage change is recorded, indicating the nucleotide has been incorporated and the base is called This process happens simultaneously in
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NGS: Applications en myco
Epidmies de C. gattii: gnotype
VGIIA: 1999-2008 Vancouver Colombie britannique, formes pulmonaires VGIIC: 2007 Etat de Washington, Oregon virulence + ++
Origines de lpidmie? Modifications gntiques lorigine de la virulence?
Engelthaler DM, Hicks ND, Gillece JD, Roe CC, Schupp JM, Driebe EM, et al. Cryptococcus gattii in North American Pacific Northwest: Whole-Population Genome Analysis Provides Insights into Species Evolution and Dispersal. mBio. 2014 Jul
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NGS: Applications Squenage gnome complet 118 souches (Illumina)
37 Engelthaler DM, Hicks ND, Gillece JD, Roe CC, Schupp JM, Driebe EM, et al. Cryptococcus gattii in North American Pacific Northwest: Whole-Population Genome Analysis Provides Insights into Species Evolution and Dispersal. mBio. 2014 Jul
NGS: Applications Souche VGIIA sud amricaines et nordamricaines trs proches
NGS: Applications Origine sudamricaine des VGIIC nordamricaines?
40 Engelthaler DM, Hicks ND, Gillece JD, Roe CC, Schupp JM, Driebe EM, et al. Cryptococcus gattii in North American Pacific Northwest: Whole-Population Genome Analysis Provides Insights into Species Evolution and Dispersal. mBio. 2014 Jul
NGS: Applications Gne de virulence? Variant VGIIA expression +++ des gnes du cluster 3. Contient collagnase => facteur de virulence pulmonaire? (idem aspergillus)
