In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer’s field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

Background The fall armyworm (FAW), Spodoptera frugiperda; J.E. Smith (Lepidoptera: Noctuidae), is now an economically important pest that causes huge losses to maize productivity in sub-Saharan Africa. Variations in sub-population genetics and the processes of rapid adaptation underpinning the invasion remain unclear. For this, the genetic identity and diversity of FAW populations in Uganda were revealed by sequencing 87 samples (collected across the country). Based on the partial mitochondrial cytochrome oxidase I (COI) gene polymorphisms, we further examined the mitochondrial haplotype configuration and compared the FAW in Uganda with sequences from other parts of the world. The molecular target for organophosphate and carbamate resistance, acetylcholinesterase, was also investigated. Results Analysis of the partial COI gene sequences showed the presence of both rice (predominant) and corn strain haplotypes, with a haplotype diversity of 0.382. Based on the COI marker, pairwise difference distribution analyses, and neutrality tests, showed that the FAW populations in Uganda and the rest of Africa are evolving neutrally, but those in America and Asia are undergoing expansion. Our findings support observations that invasive FAW populations throughout the rest of Africa and Asia share a common origin. Sequencing of the S. frugiperda ace-1 gene revealed four amino acid substitutions, two of which (A201S and F290V) were previously shown to confer organophosphate resistance in both S. frugiperda and several other insect species. The other two previously reported new variations in positions g-396 and g-768, are presumed to be related to the development of insecticide resistance. Conclusions This research has increased our knowledge of the genetics of FAW in Uganda, which is critical for pest surveillance and the detection of resistance. However, due to the low gene polymorphism of COI, more evolutionary studies incorporating the Spodoptera frugiperda whole-genome sequence are required to precisely understand the FAW population dynamics, introduction paths, origin, and subsequent spread.

In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This single-step closed system delivers purified DNA with no cross contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into base called data locally in real time, removing the need for high specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and set up.To evaluate and validate capability of the system for unbiased pathogen identification by realtime sequencing in a farmer’s field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or completely destroy cassava crops. 800 million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint on its production (https://cassavavirusactionproject.com). Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus resistant or replacement planting.This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis including sample collection, processing and provisional sequencing results was complete in under 4 hours. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

The fall armyworm (FAW), Spodoptera frugiperda; J.E. Smith (Lepidoptera: Noctuidae), has become a newly established economically important pest that causes huge losses to maize productivity in sub-Saharan Africa. In 2017, a survey was conducted to collect S. frugiperda specimens across 11 agro-ecological zones in Uganda. Cytochrome oxidase subunits 1 (CO1A and CO1B) and triose-phosphate isomerase (Tpi) partial gene segments of FAW were sequenced and subjected to comparative genomic analyses, to elucidate genetic identity, diversity and determine the existence of corn sub-haplotypes, distribution, and likely origin of invading populations. Results revealed the presence of both rice and corn strains (haplotypes h4 and h1), with rice strain being more predominant. The analyses of both mitochondrial and nuclear Tpi marker sequences showed that the FAW host strain haplotypes from Uganda were identical to those found in Florida, three Asian nations (China, India, and Pakistan) and Australia. A wide distribution of both strains was observed across all agro-ecological zones in Uganda. Comparative genomic analyses showed strain identification concordant results between CO1A and CO1B partial gene markers. Upon comparison with Tpi marker result, discordances in discrimination of FAW strains were observed. The study findings imply that Florida, China, India and Pakistan are possible centers of origin of invasion and support a possibility of multiple introductions into Uganda and the region. There is therefore a need for in-depth characterization of FAW populations in the region and other African nations to better understand its genetics and mechanisms of invasion.