A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

Abstract

Background Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. Results The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest 2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. Conclusions Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

Stochastic Expression of Invasion Genes in Plasmodium falciparum Schizonts

“…1d, top left inset). Lastly, majority of the sequenced 1-cell transcriptomes were estimated to be between and around 40 to 44 hours post invasion (HPI) (see below) by correlation to a high resolution bulk 3D7 reference transcriptome 43 (Fig. 1e), thus con rming successful isolation and ampli cation of late asexual stages.…”

The mechanism of artemisinin resistance of Plasmodium falciparum malaria parasites originates in their initial transcriptional response

“…These samples were collected during a large clinical treatment trial (TRACII) carried out from 2016 to 2018 that also characterized the spread of artemisinin resistance in the GMS(2). We isolated total P. falciparum RNA from the patients' blood samples and performed both DNA microarray and Next Generation Sequencing (RNA-seq) analysis, as previously described (40). Overall, we analyzed 577 samples collected at study enrollment to characterize the baseline transcriptomic profiles (baseline, (bl) 0hr sample set) (Fig.…”

The mechanism of artemisinin resistance of Plasmodium falciparum malaria parasites originates in their initial transcriptional response.

“…These samples were collected during a large clinical treatment trial (TRACII) carried out from 2016 to 2018 that also characterized the spread of artemisinin resistance in the GMS 2 . We isolated total P. falciparum RNA from the patients' blood samples and performed both DNA microarray and Next Generation Sequencing (RNA-seq) analysis, as previously described 40 . Overall, we analyzed 577 samples collected at study enrollment to characterize the baseline transcriptomic pro les (baseline, (bl) 0hr sample set) (Fig.…”

“…S1b). While the DNA microarray was used to generate the transcriptomes for all collected samples, there were su cient levels of parasite mRNA to perform RNA-seq-based transcriptome analyses for 188 and 159 of the (bl) 0hr and (tr) 6hr samples respectively 40 (Fig. S1c and S1d).…”