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

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)

If Robert KochWas to visit yourMicrobiology Lab

Would he have aFeeling ofDeja vu ?

Traditional Laboratory Diagnosis

Sequence of testing in diagnostic labs

RV 16 ReportNasopharyngeal Swab is positive for

Human Rinovirus, Adenovirus and Parainfluenza 1

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

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

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)

Disruptive technologyLeap from Research to Clinical Labs

Real Time PCR vs PCRMinION NGS vs NGS

Nanopore is a disruptive Technology

Flow Cell

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hours

NGS: The ultimate tool in epidemiological typing

Restriction FragmentLength Polymorphism

Multi Locus Sequence typing

Pulse Field Gel Electrophoresis

NGS: The ultimate tool in epidemiological typing

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

Thank You for

your attention