Low-volume Plasmodium blood sample processing protocols for untargeted transcriptomics optimized using Plasmodium knowlesi

As the importance of transcriptional variation and regulation for <i>Plasmodium</i> becomes more apparent, advances for non-<i>falciparum</i> 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 <i>Plasmodium knowlesi</i> culture diluted in whole blood as a mock <i>Plasmodium vivax</i> natural infection to compare white blood cell (WBC), rRNA and globin depletion methods and RNA-seq library preparation kits to create an optimized protocol for low-volume sample processing. Depletion techniques and library preparation kit selection are both crucial to enrich parasite RNA in low-parasite density and low-volume samples. WBC depletion methods can increase the percentage of mapped <i>Plasmodium</i> paired-end (PE) reads from 5.4 to 37.2-54.5%, depending on the method. Additionally, globin RNA and rRNA depletion significantly decreased the human globin RNA and rRNA combined gene counts up to 90%. When comparing the library preparation kits, the transcription profiles of the mRNA kits were highly correlated and enriched for protein-coding genes. The total RNA library prep kit was dominated by <i>P. knowlesi</i> rRNA, despite having the highest percentage of mapped <i>P. knowlesi</i> PE reads. Parasite sequencing output can be optimized when WBC and globin depletion are used in combination with an mRNA library preparation kit bringing untargeted <i>Plasmodium</i> transcriptomics to resource-limited settings which will lead to new insights in parasite biology.