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

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...

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Autores principales:	Catarina Bourgard, Stefanie C. P. Lopes, Marcus V. G. Lacerda, Letusa Albrecht, Fabio T. M. Costa
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e777c4b379b74114a1e6f3fe83cfb828
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spelling	oai:doaj.org-article:e777c4b379b74114a1e6f3fe83cfb8282021-12-02T13:34:57ZA suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients10.1038/s41598-021-84607-w2045-2322https://doaj.org/article/e777c4b379b74114a1e6f3fe83cfb8282021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84607-whttps://doaj.org/toc/2045-2322Abstract 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 ~ 100 pg.µL−1 of RNA, suitable for SMART-Seq Ultra-Low Input RNA library and Illumina sequencing. Unbiased coding transcriptome of ~ 4 M reads was achieved for four patient isolates with ~ 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.Catarina BourgardStefanie C. P. LopesMarcus V. G. LacerdaLetusa AlbrechtFabio T. M. CostaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Catarina Bourgard Stefanie C. P. Lopes Marcus V. G. Lacerda Letusa Albrecht Fabio T. M. Costa A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients
description	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 ~ 100 pg.µL−1 of RNA, suitable for SMART-Seq Ultra-Low Input RNA library and Illumina sequencing. Unbiased coding transcriptome of ~ 4 M reads was achieved for four patient isolates with ~ 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.
format	article
author	Catarina Bourgard Stefanie C. P. Lopes Marcus V. G. Lacerda Letusa Albrecht Fabio T. M. Costa
author_facet	Catarina Bourgard Stefanie C. P. Lopes Marcus V. G. Lacerda Letusa Albrecht Fabio T. M. Costa
author_sort	Catarina Bourgard
title	A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients
title_short	A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients
title_full	A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients
title_fullStr	A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients
title_full_unstemmed	A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients
title_sort	suitable rna preparation methodology for whole transcriptome shotgun sequencing harvested from plasmodium vivax-infected patients
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e777c4b379b74114a1e6f3fe83cfb828
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A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients

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