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

Catarina Bourgard,Fabio T M Costa,Stefanie C P Lopes,Letusa Albrecht,Marcus V G Lacerda

doi:10.1038/s41598-021-84607-w

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.

Highlights

Plasmodium vivax is a world-threatening human malaria parasite, whose biology remains elusive
The application of Whole Transcriptome Shotgun Sequencing (WTSS), known as RNA-seq, is a powerful approach to identify strain-specific patterns of gene expression associated with parasite virulence and host–pathogen interactions[31]
Eight RNA isolation approaches were tested in a panel of P. falciparum samples with a wide-ranging parasite density ( 102 to 107 per sample)

Summary

Introduction

Plasmodium vivax is a world-threatening human malaria parasite, whose biology remains elusive. The application of Whole Transcriptome Shotgun Sequencing (WTSS), known as RNA-seq (high throughput sequencing of the transcriptome), is a powerful approach to identify strain-specific patterns of gene expression associated with parasite virulence and host–pathogen interactions[31] To overcome these difficulties, a methodologic framework was developed which allowed to successfully achieve all steps from RNA isolation in a set of lower parasitemia from P. vivax Amazonian isolates to an unbiased coverage WTSS. Quantity and quality of parasite RNA as well as reproducibility of RNA-seq results are discussed here This approach enables genome-wide expression studies in P. vivax at endemic laboratories that will shed light on its pathogenicity, pinpoint resistance mechanisms, and facilitate validation of parasite stage-specific drug targets

Methods

Results

Conclusion