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A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients
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 f...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930272/ https://www.ncbi.nlm.nih.gov/pubmed/33658571 http://dx.doi.org/10.1038/s41598-021-84607-w |
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author | Bourgard, Catarina Lopes, Stefanie C. P. Lacerda, Marcus V. G. Albrecht, Letusa Costa, Fabio T. M. |
author_facet | Bourgard, Catarina Lopes, Stefanie C. P. Lacerda, Marcus V. G. Albrecht, Letusa Costa, Fabio T. M. |
author_sort | Bourgard, Catarina |
collection | PubMed |
description | 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 | Online Article Text |
id | pubmed-7930272 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-79302722021-03-05 A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients Bourgard, Catarina Lopes, Stefanie C. P. Lacerda, Marcus V. G. Albrecht, Letusa Costa, Fabio T. M. Sci Rep Article 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. Nature Publishing Group UK 2021-03-03 /pmc/articles/PMC7930272/ /pubmed/33658571 http://dx.doi.org/10.1038/s41598-021-84607-w Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Bourgard, Catarina Lopes, Stefanie C. P. Lacerda, Marcus V. G. Albrecht, Letusa Costa, Fabio T. M. A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax-infected patients |
title | 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_short | 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 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930272/ https://www.ncbi.nlm.nih.gov/pubmed/33658571 http://dx.doi.org/10.1038/s41598-021-84607-w |
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