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Microbial Genomics logo Microbial Genomics

Volume 11, Issue 11

Low-volume blood sample processing protocols for untargeted transcriptomics optimized using Open Access

This article is part of the Microbial Genomics of Eukaryotes collection

Abstract

As the importance of transcriptional variation and regulation for becomes more apparent, advances for non- 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 culture diluted in whole blood as a mock 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 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 rRNA, despite having the highest percentage of mapped 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 transcriptomics to resource-limited settings which will lead to new insights in parasite biology.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): field studies , parasitology , Plasmodium , sample processing , transcriptomics and white blood cell depletion
Funding
This study was supported by the:
  • Fonds Wetenschappelijk Onderzoek (Award PhD-SB Fellowship (1SC5522N))
    • Principal Award Recipient: ErinSauve
  • Departement Economie, Wetenschap en Innovatie (Award SOFI)
    • Principal Award Recipient: AnnaRosanas-Urgell
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2025-11-20
2025-12-16

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Low-volume Plasmodium blood sample processing protocols for untargeted transcriptomics optimized using Plasmodium knowlesi
M Gen 11, 001546 (2025); https://doi.org/10.1099/mgen.0.001546
/content/journal/mgen/10.1099/mgen.0.001546
/content/journal/mgen/10.1099/mgen.0.001546
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