Oxford-Nanopore eBook - 13

the Seq & Treat programme was started as a joint effort by
the Foundation for Innovative New Diagnostics (FIND) and
Unitaid. The goal is to evaluate the use of targeted NGS (tNGS)
for diagnosis of DR-TB in low- to middle-income countries by
generating clinical evidence to support WHO guidance for the
use of targeted sequencing for DR-TB identification.
This process involves evaluating the performance of existing
tNGS methods, with the aim of integrating selected tests into
established diagnostic workflows globally in the near future.
The programme requires a test for DR-TB to fulfil the
following criteria:

*	 >98% specificity and sensitivity for drug resistance
detection;

*	 100 -5,000 CFU/ml as the limit of detection;
*	 detection of hetero-resistance from 1-10% (equal to
10 resistant reads in 100 -1,000 reads, and 500 resistant
bacteria in 4,500);

*	 minimal cross reactivity with non-tuberculous mycobacteria (NTM); and

*	 <5% indeterminate results.
O'Grady has been working to devise a test to fulfil these criteria
using the Oxford Nanopore MinION. The MinION offers: easy
deployment globally at low cost; real-time analysis and rapid
turnaround of results; flexible sample numbers (depending on
the size of the sequencing facility and the number of samples);
and low cost-per-sample ($15 per sample at 80 samples/flow
cell, which is the output they are aiming for).
The workflow for nanopore sequencing and turnaround
involves 1 hour of DNA extraction, a 17-target PCR (2.5 hours),
library preparation (4.5 hours for many samples, using the
Ligation Sequencing Kit and PCR barcoding expansion),
MinION sequencing overnight, (although "if you have twelve
you could do this in a few hours," he says), and finally, analysis
using the EPI2ME workflow to investigate the TB resistance
profile. The 17 targets examined are associated with both
first- and second-line drug resistance. In all, the workflow time
ranges from 9-24 hours, depending on how many samples are
being run.

By the Numbers
In terms of sequencing metrics, O'Grady's team have designed
the primers to have an average read length of ~1 kb, which
typically have a quality score around 10. The mean depth
of coverage achieved is ~12,871x (range 2,382x - 28,301x).
O'Grady says that he would like the depth of coverage to be

more even across the 17 targets, but considering it took some
time to get a few of the targets working, he says, "we can live
with that."
By performing serial dilutions, the limit of detection was
determined between~10-100 cells per ml (>50x coverage),
or about 10x depth of coverage on the 10-cell equivalent.
O'Grady says he was "very pleased" that the primers were
working down to such low concentrations of DNA.

"

The MinION platform is flexible
and cost-effective and very
easily deployable.

"

To establish the test's specificity, O'Grady has used the test
on clinical sputum containing a range of mycobacterial
species and compared the results with publicly available
non-tuberculous mycobacteria (NTM) FastQ datasets. O'Grady
says that 15/17 assay targets were consistently specific for
MTB members. (the two exceptions target 16S and 23S rDNA,
O'Grady isn't too concerned about this lower specificity for
these targets.)
The resistance profile of TB samples is accurately predicted
using the EP2ME TB analysis tool which identifies SNPs in the
target amplicons known to cause drug resistance.
O'Grady says his team now plans to test their method on
about 400 well-defined sputum samples, provided by FIND,
to define clinical performance. These samples contain varying
levels of TB bacteria, different resistance phenotypes and
genotypes, as well as other bacteria.  
"We have got to see if we can find all that," he says. The test
will proceed to a global trial if performance is sufficiently
promising. n

Watch Justin's full talk

https://nanoporetech.com/justinogrady

...or read more about O'Grady's research on metagenomic
sequencing for rapid investigation of bacterial lower
respiratory tract infections:  https://nanoporetech.com/
ogradypublication

clinicalomics.com

13
https://www.nanoporetech.com/justinogrady https://www.nanoporetech.com/ogradypublication http://www.clinicalomics.com
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