Will the next big “disruption” in clinical microbiology be Nanopore sequencing technology? I believe this is entirely possible, but there is still work to do…

The last big “disruption” to take place in clinical microbiology laboratories was the introduction of MALDI-TOF for organism identification. From proof of concept to commercial introduction of this technology took a couple of decades. Major disruptions actually take a lot of fine tuning and polishing…

For a platform to be successful in a routine diagnostic microbiology laboratory it needs to have several key characteristics. It needs to be fast, it needs to be cost-effective (compared with existing methodology), it needs to be scalable, and it needs to perform well (good sensitivity and specificity). MALDI-TOF has achieved each of these key goals. That’s why it has been adopted, almost universally in clinical microbiology laboratories. Lots of other innovative technologies come close, but don’t quite get there…

So what about Nanopore sequencing?

Championed by Oxford Nanotech , nanopore sequencing is a unique, scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein “nanopore”. The different bases give a specific change in ionic current. The resulting signal can thus be decoded to provide the specific DNA or RNA sequence. Nanopore sequencing enables direct, real-time long-read analysis of DNA or RNA fragments.

Nanopore sequencing has several potential applications in the clinical microbiology laboratory, such as:

• Organism detection directly from clinical samples, either by 16S/18S rRNA or by a metagenomic approach
• Detection of genotypic resistance determinants
• Detection of genotypic virulence determinants
• Typing of microorganisms for infection control or public health reasons

How does Nanopore sequencing weigh up on each of the key features required to break into a routine diagnostic microbiology laboratory.

• Speed: The technology allows both base reading and bioinformatic analysis of the sequences to be performed in real-time. Depending on what is being sequenced, it is possible to get useful information from sequencing in a matter of minutes. Potentially the sequencing process can be “stopped” when the necessary information has been obtained, saving on both time and flow cell.
• Cost-effectiveness: Compared to other sequencing platforms, the start up costs are relatively low. For just a few thousand dollars, it is possible to get hold of a MinION, a few flow cells, and start “sequencing”. However other costs to consider include the bioinformatic software, and hardware to assess DNA/RNA quality and quantity. The flow cells containing the nanopores are still expensive at the moment, but the cost is decreasing. Flow cells can be washed and re-used to a certain extent, which will reduce costs. In addition, a smaller & cheaper flow cell called a “flongle” has just been released.

  

• Scalability: The ability to “barcode” the nucleic acid extracts going into the flow cell allows the processing of multiple samples simultaneously. Platforms which include multiple flow cells such as the GridION and PromethION can thus process literally hundreds and thousands of samples in the one day. The recently released LamPORE testifies to this.
• Good performance: There is a lot of validation work currently going on for Nanopore sequencing for various clinical applications, both microbiological and non-microbiological. A lot of the bioinformatic pipelines that would facilitate commercialisation of Nanopore sequencing are still in development. This will take time. Metagenomic approaches to organism identification from clinical samples using Nanopore sequencing are potentially very attractive. The issues of filtering pathogenic DNA out from the human DNA are currently being addressed.

The exciting thing about this technology is that it seems to be improving very quickly. One of the main issues historically with nanopore sequencing was the fidelity/accuracy of the base calling. However recent improvements in the nanopore design and the reading software have improved this dramatically.

I suspect over the next few years, routine clinical microbiology laboratories, like my own, will start looking closely at this technology to see whether it is ready for implementation in diagnostic clinical microbiology. I suspect it will have an initial role in sterile site samples and resistance genotyping, but may well extend to more routine samples in due course.

I think it is just a matter of time…

Michael

I am keen to hear from clinical microbiologists who have Nanopore sequencing in their laboratory, so I can learn from their experiences!