Global TB Drug Pipeline- The Need and the Reality- From the TB Alliance
Making TB diagnostics a practical reality - … TB diagnostics a practical reality Zamin Iqbal...
Transcript of Making TB diagnostics a practical reality - … TB diagnostics a practical reality Zamin Iqbal...
© Copyright 2016 Oxford Nanopore Technologies 2
Clinical test for a TB patient
• Take a sample (sputum) and culture it. Is it M. tuberculosis ?
• Need to know which drugs will work
• Mix bug and drug, and see if it grows or dies
Slow ~ 2 months Multiple rounds of culture and testing
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Pre-MinION timeline for a DNA-based approach
Get a sample, decode the genome once• Drug resistance is encoded in the DNA• Can look at strain/transmission
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Mykrobe Predictor
• Determine species (in PHE workflow for accreditation) and antibiotic resistance.• Performance for TB matches commercial molecular assays• Sensitivity/specificity of 82.6% / 98.5 % (N=1600 Illumina data)• £5million grant (WT/Gates) to sequence/phenotype 100,000 Mtb – push up sensitivity
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A long way from a same-day diagnostic
1. The biggest barrier is culture2. Sequencing time/MiSeq batch effects3. Human cells and nasal flora dominate sputum
2 weeks of culture 1-2d seq
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A long way from a same-day diagnostic
1. The biggest barrier is culture Sequence from sputum2. Sequencing time/MiSeq batch effects3. Human cells and nasal flora dominate sputum
2 weeks of culture 1-2d seq
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Sequencing TB direct from sputum
Depth of coverage of Mtb0 50 100 150
Watch out for preprint on BiorxivAntonina Votintseva
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A long way from a same-day diagnostic
1. The biggest barrier is culture Sequence from sputum2. Sequencing time/MiSeq batch effects3. Human cells and nasal flora dominate sputum
2 weeks of culture 1-2d seq
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A long way from a same-day diagnostic
1. The biggest barrier is culture Sequence from sputum2. Sequencing time/MiSeq batch effects MinION3. Human cells and nasal flora dominate sputum Read Until
2 weeks of culture 1-2d seq
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Removing contamination via Read Until
3. Nanonet basecalls fragmentsMap fragments to see if TBReturn message to ReadUntil
Using 10-minute prep for 1D reads with R9 flow cell
1. Discard first 100 events2. Take first 250 events and send to our ReadUntil server
4. Reject read while still in pore if not TB
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Results 1: in silico reanalyse data from an R9 run (human+TB)
Send events to server, base call, map, decide, send message back- all in ~10% of time it takes for read to pass through pore. (but this is biased..)
Min 1st Qu Median Mean 3rd Qu Max
Round trip 0.00 1.23 1.26 1.22 1.33 15.9
Read time in pore
2.0 7.08 11.92 20.20 22.02 4004
% 0 5.5 10.5 13 17 375
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Results 2: live run this week
Mix of human DNA (fragment size >4kb) and M. tuberculosis (~1kb)
~3 or 4:1 human:TB
See if we can keep up with the flow cell, and reduce the amount of human reads
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Human reads
Before we turned on Read Until
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Human reads
Befire Read Until With ReadUntil
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Before Read Until With ReadUntil
M. tuberculosis reads
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On the verge of a revolution in TB healthcare!
Every component needs work, but..
• 0.5-1 hour pre-step in Cat3 (decontam, heat-kill)• 2.5 hours DNA extraction • 10 minutes sample prep• ?6 hours of sequencing with in-pore filter (depends on %Mtb)• Seconds of computation
From sputum to diagnosis in <1 day
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Acknowledgements
Antonina Votintseva (TB extraction protocol)
Louise Pankhurst, Luke Anson (MinIONing)
Phelim Bradley, Carlos del Ojo Elias (RU server)
Matt Loose
Derrick Crook
ONT: Tim Massingham, Rich Ronan, Olly Hartwell