Successful Profiling of Plasmodium falciparum var Gene Expression in Clinical Samples via a Custom Capture Array
AuthorStucke, Emily M.
Hodges, Theresa K.
Koné, Abdoulaye K.
Tangara, Bourama M.
Tallon, Luke J.
Zhou, Albert E.
Laurens, Matthew B.
Doumbo, Ogobara K.
Plowe, Christopher V.
Thera, Mahamadou A.
Travassos, Mark A.
Silva, Joana C.
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Abstractvar genes encode Plasmodium falciparum erythrocyte membrane protein- 1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche’s SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional .4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis.
DescriptionThe article processing charges (APC) for this open access article were partially funded by the Health Sciences and Human Services Library's Open Access Publishing Fund for Early-Career Researchers.
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Identifier to cite or link to this itemhttp://hdl.handle.net/10713/21174
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