Characterising Mycobacterium tuberculosis with nanopore technology

Applying nanopore sequencing for analysis of drug-resistance

Andrey described how he first heard about Oxford Nanopore Technologies when it was founded in 2005 and, from then on, was always curious about the technology. Highlighting the accuracy of the recent Kit 14 chemistry, he first used the MinION device to perform whole-genome sequencing of Mycobacterium tuberculosis cultures, before making use of targeted techniques to sequence both cultures and sputum research samples with nanopore technology. With the workflows Andrey has developed, he has helped implement the use of nanopore sequencing for the surveillance of drug-resistant TB in SRL Gauting.

Andrey highlighted how, with the targeted nanopore sequencing workflow, he and his colleagues can easily sequence up to 94 samples simultaneously on a single MinION Flow Cell, producing more than 50x depth of coverage per sample. Andrey commented that long nanopore reads, as well as the good coverage and accuracy of nanopore sequencing, are advantageous for the analysis of antimicrobial resistance genes in M. tuberculosis. Long reads are particularly beneficial in this application as full-length amplicons can be sequenced, and the long reads can span and delineate complex regions of genes to help identify mutations that may lead to drug resistance. However, for Andrey, the biggest advantage of Oxford Nanopore sequencing devices over other sequencing platforms is the simplicity and the minimal costs of nanopore sequencing workflows, which he has found to be a huge benefit for his training programme.

Nanopore sequencing training programme

Andrey and his colleagues have set up training courses which they have run in laboratories across Central Asian countries to train individuals to perform TB sequencing with nanopore technology to track drug-resistance. Andrey highlighted that the minimal cost, with the ease of shipping and installation of the MinION sequencing device especially, is hugely beneficial for these laboratories and the training service. A further key advantage of the MinION device, Andrey highlighted, is the minimal power required and the ability to pause your sequencing run. Quite often, power outages can occur in the laboratories; with other sequencing platforms, Andrey explained, the sequencing run would be lost along with the power. With a MinION plugged into a laptop, sequencing can simply be paused or stopped until power is restored. Andrey also noted that the MinION does not require precise climate control in the laboratory and can sequence in non-air-conditioned rooms, further making them suitable for use in higher ambient temperatures in locations across Central Asia.

Andrey further explained that with the minimal cost and simplicity of the nanopore sequencing device and workflows, this is the ‘most advanced and most useful technology’ for their TB sequencing workflow as well as for the training programme. He described how it is much easier to teach people how to use nanopore sequencing devices compared to other sequencing methods, due to the simplicity of the workflows, even when training those who do not have prior experience of using sequencing technology. Therefore, the simplicity of nanopore sequencing is a huge advantage, improving the accessibility of DNA sequencing to laboratories and enabling more people to perform sequencing for research despite their level of prior sequencing experience.

‘Start as soon as possible’ with nanopore sequencing

For individuals wanting to get started with nanopore sequencing in their laboratories, Andrey recommends to just ’start as soon as possible’. The knowledge and experience will follow. With the ease of initial set-up and relative simplicity of the library preparations for sequencing, Andrey reiterated how 'it’s easier [and] more straightforward’ to start sequencing using Oxford Nanopore devices compared to traditional sequencing methods. His advice was similar for those who wish to get started in microbiological research in general; he emphasised that due to the recent advances in sequencing technology, this is the best time to get into the field ’to improve our lives’ across every area of research, from food to health.

‘We are living in the time [of] a revolution in biotechnology’

The next steps Andrey has planned for his laboratory is to move away from other sequencing platforms and to focus on nanopore sequencing due to the many benefits, ‘starting from [the] simplicity of use and costs’. For the future of Oxford Nanopore Technologies, Andrey is most excited to see the development of streamlined and simplified sequencing workflows, from library preparation through to data analysis. He stated his belief that ’it has really great potential for clinical diagnostics’* and ’it’s just a matter of time’ until the bioinformatics is simplified to a single click of a button for a full report to be produced after a couple hours of sequencing.

*Oxford Nanopore Diagnostics are developing a TB drug resistance (TB-DR) test for clinical use as described in the WHO rapid communication (July 2023).  The end-to-end workflow, inclusive of reagents, GridION device and software will be manufactured under ISO 13485 and will be submitted for registration with global regulatory bodies (FDA, CE (IVDR), CDSCO).