Members of the MalariaGEN Resource Centre, working at the Wellcome Trust Sanger Institute, are actively developing methods to overcome the technical challenges involved in sequencing parasite DNA isolated directly from the blood of malaria patients. Working with these field samples poses many challenges including high-levels of human DNA contamination, insufficient volumes, and degradation during transit.
Ultimately, the goal is to develop methods that, when coupled with advances in sequencing platforms like Illumina, result in the production of good quality parasite sequence data, even from samples with low parasite densities. As they become available, we will share information about the methods and protocols here.
- Collection of dried blood spots (DBS) of Plasmodium-infected whole blood
- Leucocyte depletion of 2.0ml of Plasmodium-infected whole blood using MN2100ff cellulose columns
There are several advantages of using dried blood spots to collect parasite samples for DNA sequencing:
- Uses small volumes of blood – this makes collection from sick patients easier, and also simplifies storage and transport.
- No cold chain required – once spotted, the blood can be kept at room temperature.
- Low cost – minimal training and materials needed.
Despite these advantages, there are several obstacles that have limited the use of dried blood spots in the past, namely inconsistencies in materials, procedural variability, and issues with sample tracking and archiving. For example, blood spots of different sizes leads to variable DNA yields; spot card thickness or type affects blot spread which can create inconsistencies across different sites; extraction efficiency, storage and transit conditions can lead to sample loss or contamination; and inconsistencies in labelling can lead to processing errors.
To help overcome these challenges, we’ve developed a new protocol for our partners who are collecting dried blood spots. The protocol was tested and validated in 2016, as part of a pilot field study in Ghana.
Download a step-by-step guide that includes guidance for:
- Preparing DBS kits for collection
- Barcoding DBS for easy tracking and archiving
- Consistently collecting fixed volume, evenly-sized blood spots
- Storing and transporting DBS
A primary obstacle to whole genome sequencing (WGS) parasite DNA directly from infected whole blood is the overwhelming contamination of human leucocyte DNA (typically >99% of the sample). This challenge can be overcome with methods like hybrid RNA baiting and magnetic separation, however these methods aren’t always amenable to the field. Cellulose powder columns are a low-cost, easy-to-assemble alternative for malaria patient sampling in remote field sites that lack extensive lab facilities.
This protocol involves a leucodepletion cellulose column method that uses MN2100ff as an alternative to Sigma® (Whatman®) CF11 cellulose, which is no longer commercially available. The efficacy of leucodepletion, and ease of blood sampling and filtration was tested in the laboratory and during a pilot field study carried out in 2015. This protocol is provided pre-publication, and is subject to change.