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Tom Nieto: How nanopore sequencing is changing HLA typing for renal transplants in low income countries

Spotlight session: Tom outlined that his application is one particularly suited to expansion to the developing world, where tissue typing labs simply do not exist. But more than that, how can we take portable tissue typing into clinical practice, and how do we validate it?

Tom explained that kidney disease affects 1 in 10 adults worldwide, and that transplantation is the best possible form of treatment for kidney disease, improving quality and length of life of the patient versus the alternative, haemodialysis. Safe transplantation requires tissue matching to avoid rejection, and the better the tissue match, the longer the kidney lasts before further intervention is required.

To accommodate this need for rapid tissue typing for organ matching, Tom introduced the “mark one” tissue typing laboratory in a suitcase, featuring such equipment as the miniPCR, MinION, and a laptop to which just pipettes and DNA could be added. Tom then asked whether such a setup could be validated quickly in Birmingham, which hosts the largest solid organ transplant system in the UK. Once validated, the equipment could be taken on the road with Transplant Links Community, a charity which takes transplantation to the developing world – in particular Ghana, the Caribbean and Papa New Guinea. In these regions, Tom explained, the problem is not a lack of patients or clinicians, merely that the labs for them to use don’t exist. Tissue samples, instead of being processed in-country, have to be sent back to the UK to establish whether they are a match to the patient or not. This clearly isn’t a sustainable method and demonstrates an urgent need for technology near the location of tissue sampling.

In more detail, the process for nanopore sequencing of the tissue samples involved use of the Ligation Sequencing Kit with barcoding, before using Porechop for demultiplexing, alignment with minimap and samtools, and finally HLA-PRG-LA for HLA typing. All software is open-source and so easy for other users to replicate.

For the pilot scheme, Tom described their analysis of 11 samples from kidney donors, extracting DNA from patient blood and typing with short-read sequencing, SSP (sequence-specific primer) techniques, and nanopore long read sequencing. Tom detailed that traditional SSP methods type to single field accuracy and overall the process carrries approximately 1% error – not in least because technicians must manually transfer typing results across to a large spreadsheet, often late at night.

While the short-read sequencing yielded 4-field accuracy, MinION comfortably obtained 3-field accuracy, with further optimisation yet to be done. Combined with this result, MinION also provides the advantage of being suitable for overnight provision of results, taking just 9 hrs in comparison to significantly extended timelines for short read assays. This rapid turnaround also brings closer the possibility of deceased organ transplant.

To conclude, Tom listed the advantages of nanopore sequencing for HLA typing for solid organ transplant: improved access to typing globally; removing logistical barriers to renal transplantation; lower costs; faster results; more robust protocols; and greater accuracy that commonly-used SSP techniques.

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