A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples
| Autor: | Grennady Wirjanata, Rob W. van der Pluijm, Arjen M. Dondorp, Lei Zhu, Mehul Dhorda, Jaishree Tripathi, Michal Kucharski, Sourav Nayak, Zbynek Bozdech |
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| Přispěvatelé: | Graduate School, AII - Infectious diseases, APH - Aging & Later Life, APH - Global Health, APH - Quality of Care, Intensive Care Medicine, School of Biological Sciences |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Plasmodium lcsh:Arctic medicine. Tropical medicine lcsh:RC955-962 Plasmodium falciparum 030231 tropical medicine RNA preservation RNA-Seq High-throughput Computational biology Biology Southeast asian Specimen Handling lcsh:Infectious and parasitic diseases Transcriptome 03 medical and health sciences 0302 clinical medicine parasitic diseases Humans Medicine [Science] lcsh:RC109-216 Whole Transcriptome Analysis Malaria Falciparum RNA Extraction Messenger RNA Gene Expression Profiling Methodology High-Throughput Nucleotide Sequencing RNA biology.organism_classification RNA extraction Malaria Blood 030104 developmental biology Infectious Diseases Parasitology RNA-seq RNA Protozoan Whole transcriptome analysis |
| Zdroj: | Malaria Journal, Vol 19, Iss 1, Pp 1-16 (2020) Malaria journal, 19(1):363. BioMed Central Malaria Journal |
| ISSN: | 1475-2875 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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