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

• Published in: Malaria Journal
• Main Authors: Michal Kucharski, Jaishree Tripathi, Sourav Nayak, Lei Zhu, Grennady Wirjanata, Rob W. van der Pluijm, Mehul Dhorda, Arjen Dondorp, Zbynek Bozdech
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2020
• Subjects: Whole transcriptome analysis; RNA extraction; RNA preservation; Plasmodium; RNA-seq; High-throughput; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1186/s12936-020-03436-w; https://doaj.org/article/e0193e090d314daeb5b61309fb0d286c

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 ...