Customer Spotlight – The Old Fight is New: Tackling Malaria with Next-Generation Sequencing

In your opinion, what is a major challenge in genetically characterizing malaria parasites and how does your research address this?

BG: The main issue we faced was the tradeoff between throughput, number of targets, and sensitivity. Sensitivity is particularly challenging because of the sample quantity. We start with a spot of blood on filter paper so there’s not a lot of material in general, and most of the genetic material is from the human host. To compound the issue, there are often multiple strains of the parasite in a single infection and if you miss some of them, you’re not necessarily getting the right answer. There are layers of complexity- the parasite DNA is the minority of the genetic material, we want to capture the variants of the pathogen DNA, and we’re attempting to answer multiple questions about drug resistance, diagnostic resistance (evading rapid diagnostic tests via a gene deletion), and transmission epidemiology. Our team was looking for an efficient and comprehensive solution to optimize on time and cost but not compromise on the quality of the data. We spent a couple of years researching and testing different technologies, but we couldn’t find one that worked for both sensitivity and throughput until we met Paragon Genomics. After we kicked off a pilot project with Paragon, we were very impressed with the quality of the preliminary results. Once we established proof of principle and saw we could consistently generate quality data with challenging samples, our team wanted to expand the panel, scale up and apply this method at different sites globally. Customization was a key highlight. We went through many iterations of design, and the Paragon team was very open to working with us and able to get us what we needed in terms of a comprehensive and modular panel. The result is that now we have a method that does exactly what we need. And it is robust enough that the panel meets the needs of other labs and other countries.

Are there limitations of current gold standards or other methods, and how did Paragon Genomics’ custom targeted sequencing approach help with that?

BG: The primary limitation of other methods we have evaluated is sensitivity- we’ve tried FLASH (CRISPR-based method), which worked to some extent. However, the design process was difficult, sensitivity was limited, and the chemistry wasn’t robust- it was difficult for us to understand why certain targets were working better than others. Our lab also tried using molecular inversion probes, which had UMIs and allowed for overlapping segments in the same tube, without worrying about primer dimers. Similar to Paragon’s chemistry, it allowed us to design for many targets, but the main limitation was sensitivity. There was essentially only a single cycle for capture, compared to the 10-15 cycles for multiplex PCR (mPCR). In the past, when I was working with microsatellites, I developed my own homebrew mPCR and designed my own primers, but it was difficult to expand the number of targets. And because we had such a robust solution out-of-the-box with Paragon, it was much more efficient for us to continue with a proven strategy.

What do you see as the biggest advantages of using a custom targeted NGS panel for malaria research or diagnosis, and how does your panel maximize those benefits?

BG: The biggest advantage is probably the reliability of the workflow. Maybe there are other methods out there that are as simple but there’s something to be noted about the consistent data quality from Paragon’s chemistry. We haven’t needed to repeat many experiments or throw out data from many samples, which happened more frequently with the other methods we tried. The high uniformity of balance between targets and samples and high sensitivity have been big advantages. We’re working on automation right now, and hope that it will make processing a large number of samples even easier.

How do you see NGS panels evolving in the field of malaria research and clinical practice and how do you think your panel will contribute to that evolution?

BG: We want to continue to have easy and small panels for existing applications. We’ve also been thinking about something called therapeutic efficacy studies- when we evaluate if a malaria drug works, we treat patients and then follow them for many weeks. But if a patient comes back a few weeks later with a reinfection, you need to know if the infection is the original infection (indicating they failed therapy) or if it’s an entirely new infection, indicating the drug worked well and the patient happened to get infected again. We need to identify if the parasites are the same or different. It’s been difficult because of the cumbersome methods in the past, but amplicon sequencing would be very useful in this application, particularly given the high sensitivity to detect most of the parasites present and the ability to simultaneously evaluate a number of diverse targets. As we think about leveraging the current panel and tailoring it for specific therapeutic efficacy studies, we want to statistically curate a subset of the panel (e.g. ~20 targets) for the most efficient sequencing. Our team has also been thinking about investigating interesting biological questions like how immunity develops. We want to sequence those malaria targets of the human immune system where genetic variation exists and examine associations with infection outcomes in our cohort studies and the associated immune response.