transforming clinical microbiology by next generation sequencing
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Transcript of transforming clinical microbiology by next generation sequencing
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Tranforming Clinical Microbiology by MinION NGS
Prof Ashok Rattan,Prof & Head,
Dept. of Microbiology, Immunogenetics & Molecular Biology,Mahatma Gandhi University of Medical Sciences and Technology,
Jaipur
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Sequence of talk
1. Current problem with laboratory diagnosis2. Role of Rapid diagnosis3. Sequencing methods4. What can sequencing do ?5. Clinical application of NGS
1. Diagnosis2. AST3. Epidemiology
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Number of new microbial species identified (1979 – 2012)
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If Robert KochWas to visit yourMicrobiology Lab
Would he have aFeeling ofDeja vu ?
Traditional Laboratory Diagnosis
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Sequence of testing in diagnostic labs
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RV 16 ReportNasopharyngeal Swab is positive for
Human Rinovirus, Adenovirus and Parainfluenza 1
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The First Choice Diagnostic Laboratory
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ABI 3700
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Next Generation Sequencing
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Strategies for Next Generation Sequencing
1. Whole Genome Sequencing (WGS)100+ Gb sequencing
2. Whole exome sequencing (WES)Capture all (200,00) exons & sequence themLess sequencing & analysis (38 Mbs)
3. Target sequencingCapture regions of interest & sequence them
4. Transcriptone sequencing (RNAseq)What is being actively transcribed in the cell of interest20,000 protein coding genes
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Microbial WGSBacterial genome contains 1 – 6 Million bp encodes for 3000 genesHuman genome contains 3.3 billion bp and encodes 25000 genes
More than 1500 genomes from over 200 different microbes are available
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Hospital Acquired Infection BioDetection System
Ion Torrent 3rd generation sequencer
• Simultaneously detects 17 bacterial pathogens– Acinetobacter baumannii– Acinetobacter pittii,– Acinetobacter calcoaceticus,– Clostridium difficile – Escherichia coli, – Enterococcus faecalis– Enterococcus faecium– Enterobacter aerogenes,– Enterobacter cloacae – Klebsiella oxytoca, – Klebsiella pneumonia, – Proteus mirabilis, – Pseudomonas aeruginosa,– Staphylococcus aureus, – Staphylococcus epidermidis,– Staphylococcus saprophyticus– Staphylococcus haemolyticus
• Simultaneously detects the 17 families of resistance genes– NDM (beta lactamase), – OXA (beta lactamase),– KPC (beta lactamase), – IMP (beta lactamase), – CARB (beta lactamase), – CTX-M (beta lactamase), – TEM (beta lactamase), – SHV (beta lactamase), – PER (beta lactamase), – VEB (beta lactamase), – VIM (beta lactamase), – mec A (methicillin resistance), – van A (glycopeptide resistance), – erm A (macrolide/lincosamide resistance) – mex A (multidrug resistance efflux operon)– aac (aminoglycoside resistance)– aad (aminoglycoside resistance)– ant (aminoglycoside resistance)– sul 1 (sulphonamide resistance)
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Disruptive technologyLeap from Research to Clinical Labs
Real Time PCR vs PCRMinION NGS vs NGS
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Nanopore is a disruptive Technology
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Flow Cell
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hours
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NGS: The ultimate tool in epidemiological typing
Restriction FragmentLength Polymorphism
Multi Locus Sequence typing
Pulse Field Gel Electrophoresis
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NGS: The ultimate tool in epidemiological typing
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Nepalese Origin of Cholera In HaitiClinic Micro Infect 2012; 18: E 158 – E 163
• Jan 2010 Major Earth Quake in Haiti• Oct 2010 UN Peacekeeping force from Nepal• Based in Mirabalais camp• 5 days later, outbreak • V cholerae serogroup O1, serotype Ogawa, biotype el
tor• > 500,000 infections• > 7,000 deaths• Climate factors, rising temperature, earth quake
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Thank You for
your attention