next generation sequencing in clinical diagnosis

2. Sequence of talk
1. Current problem with laboratory diagnosis
2. Role of Rapid diagnosis
3. Sequencing methods
4. What can sequencing do ?
5. Clinical application of NGS
1. Diagnosis
2. AST
3. Epidemiology

3. •
Number of new microbial species identified (1979 – 2012)

5. If Robert Koch
Was to visit your
Microbiology Lab
Would he have a
Feeling of
Deja vu ?
Traditional Laboratory Diagnosis

6. Sequence of testing in diagnostic labs

15. RV 16 Report
Nasopharyngeal Swab is positive for
Human Rinovirus, Adenovirus and Parainfluenza 1

18. The First Choice Diagnostic Laboratory

19. •

20. ABI 3700

21. •
Next Generation Sequencing

22. •

23. Strategies for Next Generation Sequencing
1. Whole Genome Sequencing (WGS)
100+ Gb sequencing
2. Whole exome sequencing (WES)
Capture all (200,00) exons & sequence them
Less sequencing & analysis (38 Mbs)
3. Target sequencing
Capture regions of interest & sequence them
4. Transcriptone sequencing (RNAseq)
What is being actively transcribed in the cell of interest
20,000 protein coding genes

24. Microbial WGS
Bacterial genome contains 1 – 6 Million bp encodes for 3000 genes
Human genome contains 3.3 billion bp and encodes 25000 genes
More than 1500 genomes from over 200 different microbes are available

26. 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)

27. Disruptive technology
Leap from Research to Clinical Labs
Real Time PCR vs PCR
MinION NGS vs NGS

29. Nanopore is a disruptive Technology

32. Flow Cell

38. •

40. hours

43. NGS: The ultimate tool in
epidemiological typing
Restriction Fragment
Length Polymorphism
Multi Locus Sequence typing
Pulse Field Gel Electrophoresis

44. NGS: The ultimate tool in
epidemiological typing

45. Nepalese Origin of Cholera In Haiti
Clinic 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