A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients

Author(s)

Bourgard, Catarina ^[1] ; Lopes, Stefanie C. P. ^{[2, 3]} ; Lacerda, Marcus V. G. ^{[2, 3]} ; Albrecht, Letusa ^{[1, 4]} ; Costa, Fabio T. M. ^[1]

Affiliation

^[1] Univ Campinas UNICAMP, Inst Biol, Dept Genet Evolut Microbiol & Immunol, Lab Trop Dis, Campinas, SP - Brazil

^[2] Fundacao Oswaldo Cruz FIOCRUZ, Inst Leonidas & Maria Deane, Manaus, AM - Brazil

^[3] Fundacao Med Trop Dr Heitor Vieira Dourado FMT HV, Gerencia Malaria, Manaus, AM - Brazil

^[4] Fundacao Oswaldo Cruz Fiocruz, Inst Carlos, Curitiba, PR - Brazil

• Document type: Journal article
• Source: SCIENTIFIC REPORTS; v. 11, n. 1 MAR 3 2021.
• Web of Science Citations: 1

Abstract

Plasmodium vivax is a world-threatening human malaria parasite, whose biology remains elusive. The unavailability of in vitro culture, and the difficulties in getting a high number of pure parasites makes RNA isolation in quantity and quality a challenge. Here, a methodological outline for RNA-seq from P. vivax isolates with low parasitemia is presented, combining parasite maturation and enrichment with efficient RNA extraction, yielding similar to 100 pg.mu L-1 of RNA, suitable for SMART-Seq Ultra-Low Input RNA library and Illumina sequencing. Unbiased coding transcriptome of similar to 4 M reads was achieved for four patient isolates with similar to 51% of transcripts mapped to the P. vivax P01 reference genome, presenting heterogeneous profiles of expression among individual isolates. Amongst the most transcribed genes in all isolates, a parasite-staged mixed repertoire of conserved parasite metabolic, membrane and exported proteins was observed. Still, a quarter of transcribed genes remain functionally uncharacterized. In parallel, a P. falciparum Brazilian isolate was also analyzed and 57% of its transcripts mapped against IT genome. Comparison of transcriptomes of the two species revealed a common trophozoite-staged expression profile, with several homologous genes being expressed. Collectively, these results will positively impact vivax research improving knowledge of P. vivax biology. (AU)

• FAPESP's process: 12/16525-2 - Plasmodium vivax: pathogenesis and infectivity
• Grantee: Fabio Trindade Maranhão Costa
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 13/20509-5 - Analyses of the immunopathological and molecular mechanisms involved in cytoadherence of Plasmodium vivax
• Grantee: Catarina Baeta da Luz Bourgard
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 17/18611-7 - Development of new tools for search and validation of molecular targets for therapy against Plasmodium vivax
• Grantee: Fabio Trindade Maranhão Costa
• Support Opportunities: Research Projects - Thematic Grants