Tree Lab: Portable Genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa

Abstract

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.

DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

“…The potential of the Nanopore platform for sequencing human and plant pathogens in varied and challenging locations is well established (10–13). It works on the principle of nanopore sequencing of DNA strands, with read lengths of several hundred to hundreds of thousands of bases obtained in real time (29, 30).…”

“…Furthermore, most DNA sequencing platforms (eg Illumina) have high capital costs, require reliable power supplies, cold chain reagent shipping and highly trained staff. In contrast, the Oxford Nanopore Technologies platform, (Oxford, UK), determines nucleotide sequences via a compact, portable device powered using a laptop USB port, which can be operated in varied and challenging locations (10–13). This offers a potential direct-from-sample sequencing platform for settings with the highest burden of TB – following the third pillar of the WHO End TB Strategy, ‘intensified research and innovation’ (1).…”

A computational strategy for rapid on-site 16S metabarcoding with Oxford Nanopore sequencing

“…A major advantage of ONT sequencing with MinION is portability, as well as the robustness of the protocols involved; the latter is not a minor feature: in recent years, several campaigns have been performed to test portable equipment and optimize reagents and protocols for molecular biology and sequencing library preparation, so that reliable operating procedures can be conducted in the field, including working in difficult conditions (Quick et al, 2016;Castro-Wallace et al, 2017;Arwyn et al, 2019). Indeed, with an affordable budget, for equipment and reagents, it is possible (and has been done, by more and more scientists and researchers) to perform a full investigation in the field, from sampling to data analysis (Maestri et al, 2019;Boykin et al, 2019). As for the latter, the approach outlined and validated in this study is specifically designed for rapid and affordable (here meant as low computational requirements, in terms of hardware and software, but also time and energy consumption) 16S metabarcoding, operated directly in situ, for environmental investigations, e.g.…”

Genetic Variation May Have Promoted the Successful Colonization of the Invasive Gall Midge, Obolodiplosis robiniae, in China

“…Genomic DNA was extracted from the entire O. robiniae body following the instructions described by Zhou et al (2007) and stored at −20 • C until needed. The 14 microsatellite loci (W3, W5, W8, W29, W31, W33, W35, W41, W46, W82, W83, W116, W126, and W132) developed by Yao et al (2015) were initially selected to analyze the genotypes of 20 individuals per collection site (the exception being Zhengzhou, for which 16 individuals were analyzed) ( Table 1).…”