Sequencing Methods


Despite the technical challenges of deploying sequencing to field laboratories, we have previously developed portable sequencing for Ebola and Zika for deployment in Guinea, Sierra Leone and Brazil[3,4,12]. Building on these experiences, we will develop and field-test a grab and go, integrated sample preparation and library generation workflow for in-field viral sequencing including all training materials and validated standard operating procedures (SOPs). Due to the system requirements we will use the pocket-sized and inexpensive Oxford Nanopore MinION sequencer but it would be compatible with other platforms. The emphasis will be on portability and suitability for resource-limited environments. Years 1-3 will focus on the development and field testing of the workflow in Sierra Leone where we and our collaborators have relevant biological samples stored. In years 4 and 5 we will deploy to remote sites either with ongoing outbreaks or where significant sample collections exist but sequencing technology is not readily available. We have links with researchers in Guinea, Uganda, Nigeria and Brazil that have such sample collections. This will also allow evaluation of the training materials developed as part of the program.

Develop portable sample inactivation and reagent/sample preparation

Our primary aim is to develop a system that can work alongside diagnostic laboratories embedded within epidemic responses using residual diagnostic nucleic acids. However, in some instances residual materials may not be adequate for sequencing, due to complexity, low viral loads and/or sub-optimal storage conditions. With this in mind, we will develop a portable laboratory module with the capacity to handle, inactivate and enrich (see WP2) diagnostic samples, creating an integrated solution for viral genome sequencing. Traditional approaches rely on the use of flexible film isolators or class III microbiological safety cabinets which are not portable or suitable for rapid deployment. A small portable glove box system and modular diagnostic laboratory setup was recently developed by the European Mobile Laboratories and used during the EVD epidemic[13]. We will refine this for sequencing and sample enrichment when required. Importantly we do not seek to establish a full diagnostic workflow, but to provide the capacity for limited sample manipulation or enrichment as required.

Integration of portable lab in a suitcase

Sample prep, amplification and library generation traditionally requires AC-powered equipment that lacks the portability required for deployment and may fail during power cuts. For example, thermocyclers are required for a number of steps in preparation of viral samples for sequencing. We will reduce the logistical burden of the sample preparation and the amplification/library prep process by developing a robust integrated and highly portable low-power consuming workflow, with the ultimate goal of operating completely “off-grid”. During our work on Ebola in Sierra Leone, we made extensive use of the recently developed MiniPCR machine (Amphylus), which can be battery powered and controlled by a mobile phone. Similar portable systems have been developed that all for true portability. We will develop a Pelicase deployable system with lithium battery packs charged by small, folding, solar panels, capable of powering laptops, DC powered centrifuges and portable PCR machines. We will investigate the integration of a small chiller unit to keep reagents cool, validate the use of stabilized enzymes and reagents in the workflow to reduce the dependence on a cold-chain for the transport and storage of the costly reagents.

Reducing contamination risk

Our experience has indicated that contamination with PCR products is a major concern when operating in field conditions. Traditional PCR cabinets provide a clean workspace that can be routinely decontaminated by UV irradiation but are heavy, costly, difficult to repair and not appropriate for field deployment. We will develop a lightweight, collapsible sample/reagent preparation cabinet, with UV decontamination, by combining and repurposing low cost, lightweight and highly portable components. This will be combined with robust internal contamination controls and validated SOPs to allow a quantitative determination of contamination, essential when using iSNVs to reveal transmission links between cases (detailed in subsequent WPs).