New, rapid sequencing tools for poliovirus surveillance

Swift and accurate characterization of poliovirus outbreaks is essential as we approach the global poliovirus eradication endgame. Current culture-based methods are sensitive but can be laborious.

During this webinar, Dr Alex Shaw highlighted how nanopore sequencing-based methods can be used for the detection of the virus in both stool and sewage, acting as additional tools to rapidly identify poliovirus cases and discover silent transmission of the virus in populations. Alex also discussed how highly portable nanopore sequencing devices can be used to bring viral sequencing to laboratories in countries where poliovirus strains still circulate.

Answers from Alex Shaw to your questions during the webinar:

1. Using this method, do you need to perform any library preparation?

This method does require library preparation, as our barcoded primers do not include the sequencing adaptor. Fewer steps are however required – the samples are simply pooled, and the sequencing adaptor added, and then the library is ready to go.

2. Would you get a better read coverage for each sample when you have a smaller number of samples?

Fewer samples do indeed give higher coverage, but we find that unless we are testing complex samples, we are not limited by read depth. Nanopore runs can be set to last for days, and we often only sequence for 4 hours for poliovirus detection from stool samples. Software such as RAMPART (https://artic-network.github.io/rampart/) allows real-time mapping of sequencing reads and can provide constant analysis of the read depth by both sample and by organism, so a run can simply be ended when a sufficient read depth is achieved.

3. What cell line is used for the cell culture?

Two cell lines are used: L20b for poliovirus specific culture and RD for cultivation of enteroviruses.

4. What is the limit of multiplexing with the MinION chemistry?

The number of barcodes available and potentially the depth required. We currently use 96 barcodes, allowing 96 samples to be processed, and our reduced library preparation protocol means less additional processing prior to sample pooling. Other protocols, e.g., native barcoding, also have 96 barcodes available. Some applications will require greater sequencing depth and, even with a long sequencing run, there may be insufficient data when all 96 barcodes are used.

5. Currently, COVID-19 is a global problem. Would it be possible to develop a dual target set of primers for both polio and COVID-19 viruses in sewage?

Yes, a multiplex PCR is indeed possible, although sensitivity may be an issue from sewage, and the relative abundance of each target would need to be considered to prevent one from masking the other in a dual assay if the product is being sequenced.

6. Could one use an iPad or even cell phone in place of a laptop for the analysis and informatics?

We currently use a MinION with a laptop with a GPU to provide sufficient processing speed for real-time analysis, and a cell phone or iPad would not have sufficient processing power. Another option would be to use the MinION Mk1C, which performs the sequencing and has built-in processing capabilities that can be controlled by an app.

7. What is the comparison of resources necessary for the new method compared with the traditional culture, especially in large-scale use, including RNA extraction?

The main trade off in resources is the cost of a dedicated cell culture facility versus the cost of performing RNA extraction on every sample, including both staff costs and reagents. With almost half of the cost of detection being staff costs (https://bmjopen.bmj.com/content/9/1/e023290) this becomes a complex calculation, as direct detection is quicker to perform. An aim of the Global Polio Eradication Initiative is to move towards direct molecular detection methods as we progress towards polio eradication.

8. What is the next step and how is the CDC global polio reference lab involved?

We are providing training in the method to GPLN (Global Polio Laboratory Network) facilities, as requested, and are performing comparisons to current alternative direct detection methods, with the aim of providing sufficient data so that an informed strategy can be established. The CDC global polio reference lab is involved in eradication on several fronts, including the development of alternative direct detection methods and of novel oral polio vaccine candidates, and the provision of genomic sequencing (https://www.cdc.gov/polio/what-we-do/index.htm).

9. Are there other samples collected, aside from stool? How are they used and what tools are used?

Sewage samples are also collected for environmental surveillance and, following processing to filter and concentrate the samples, these can be processed in the same manner, either through cell culture or direct detection. The sequencing results from these samples give a population-level view of circulating polioviruses.