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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\n                    <jats:italic>Plasmodium knowlesi<\/jats:italic>\n                    culture diluted in whole blood as a mock\n                    <jats:italic>Plasmodium vivax<\/jats:italic>\n                    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\n                    <jats:italic>Plasmodium<\/jats:italic>\n                    paired-end (PE) reads from 5.4 to 37.2\u201354.5%, 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\n                    <jats:italic>P. knowlesi<\/jats:italic>\n                    rRNA, despite having the highest percentage of mapped\n                    <jats:italic>P. knowlesi<\/jats:italic>\n                    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\n                    <jats:italic>Plasmodium<\/jats:italic>\n                    transcriptomics to resource-limited settings which will lead to new insights in parasite biology.\n                  <\/jats:p>","DOI":"10.1099\/mgen.0.001546","type":"journal-article","created":{"date-parts":[[2025,11,20]],"date-time":"2025-11-20T17:16:23Z","timestamp":1763658983000},"source":"Crossref","is-referenced-by-count":0,"title":["Low-volume Plasmodium blood sample processing protocols for untargeted transcriptomics optimized using Plasmodium knowlesi"],"prefix":"10.1099","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3282-4704","authenticated-orcid":false,"given":"Erin","family":"Sauve","sequence":"first","affiliation":[{"name":"Malariology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine Antwerp, 155 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