Optimized low-volume Plasmodium blood sample processing protocols for untargeted transcriptomics

As the importance of transcriptional variation and regulation for Plasmodium becomes more apparent, advances for non-falciparum species are hindered by our reliance on natural infections to study parasite biology. Untargeted transcriptomic research is also complicated by low parasite densities and high proportions of human genetic material, highlighting the need for optimized sample processing protocols. In this study, we used a P. knowlesi culture diluted in whole blood as a mock P. vivax natural infection to compare white blood cell, rRNA-, and globin depletion methods and RNA-seq library preparation kits to create an optimized protocol for low-volume sample processing. Bulk RNA-seq of P. knowlesi mock natural infection samples (5,000 parasites/ul) processed using different combinations of white blood cell depletion methods (buffy coat removal after centrifugation (cent/pipette), Plasmodipur filter (plasmo), Plasmodipur filter + MACS column (PMACS), cellulose column (cellulose)) in comparison to a no filter control, library preparation kits (mRNA Illumina / Qiagen, total RNA Qiagen) and globin/rRNA depletion methods. Please note that, since there was no ethical approval for sharing the human component, filtered parasite-only fastq files are provided as raw data and the count tables do include the human component as described in the data processing description.