Post-Genomic Approaches to Understanding Malaria Parasite Biology: Linking Genes to Biological Functions

Sexton, Anna E.; Doerig, Christian; Creek, Darren J.; Carvalho, Teresa G.

doi:10.1021/acsinfecdis.9b00093

Cited by 24 publications

(25 citation statements)

References 111 publications

(178 reference statements)

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“…Comparable proportions of unknowns have been reported in other parasite transcriptomic datasets, including myxosporeans 18,23,61 . Myxozoans have have undergone both extreme genome reduction and rapid molecular evolution and the high proportions of unknown proteins may reflect long branches that preclude resolving relationships to currently characterised proteins.…”

Section: Bryosalmonaesupporting

confidence: 58%

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease

Faber

Yoon

Shaw

et al. 2020

Preprint

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The myxozoan parasite, Tetracapsuloides bryosalmonae has a two-host life cycle alternating between freshwater bryozoans and salmonid fish. Infected fish can develop Proliferative Kidney Disease (PKD), characterised by a gross lymphoid-driven kidney pathology in wild and farmed salmonids. To facilitate an in-depth understanding of T. bryosalmonae-host interactions, we have adopted a two-host parasite transcriptome sequencing approach to minimize host contamination in the absence of a complete T. bryosalmonae genome. Parasite contigs common to both infected hosts (the intersect transcriptome; 7,362 contigs) were typically AT-rich (60-75% AT). 5,432 contigs within the intersect were annotated with 1,930 unannotatde contigs encoding for unknown transcripts. We have focused on transcripts encoding proteins involved in; nutrient acquisition, host-parasite interactions, development, and cell-to-cell communication or proteins of unknown function, establishing their potential importance in each host by RT-qPCR. Host-specific expression profiles were evident, particularly in transcripts encoding proteases and proteins involved in lipid metabolism, cell adhesion, and development. We confirm for the first time the presence of homeobox proteins and a frizzled homologue in myxozoan parasites.The novel insights into myxozoan biology that this study reveals will help to focus research in developing future disease control strategies.

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Section: Bryosalmonaesupporting

confidence: 58%

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease

Faber

Yoon

Shaw

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…As Plasmodium transcriptomic studies become more important both in the field and in the lab, larger sample numbers are being generated [32,33]. In order to increase the throughput and decrease variability (inherent to manual handling) between samples, sample processing automation should be gradually introduced in every laboratory that handles a large number of specimens.…”

Section: Discussion and Practical Considerationsmentioning

confidence: 99%

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

Kucharski

Tripathi

Nayak

et al. 2020

Malar J

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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.

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“…As Plasmodium transcriptomic studies become more important both in the eld and in the lab, larger sample numbers are being generated [33,34]. In order to increase the throughput and decrease variance (inherent to manual handling) between samples, sample processing automation should be gradually introduced in every lab that handle a large number of specimens.…”

Section: Discussion and Practical Considerationsmentioning

confidence: 99%

A Comprehensive RNA Handling and Transcriptomics Guide for High-Throughput Processing of Plasmodium Blood-Stage Samples.

Kucharski

Tripathi

Nayak

et al. 2020

Preprint

View full text Add to dashboard Cite

BackgroundSequencing 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, we present a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples that can be fully streamlined for high-throughput studies.Results We evaluated the utility of various commercially available RNA-preserving reagents in a range of storage conditions. Similarly, we compared several RNA extraction protocols and established the one most suitable for the extraction of high-quality total RNA from low-parasitemia and low-volume blood samples. Furthermore, we updated the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA. Optimization of SMART-seq2 amplification method to better suit AT-rich P. falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10ng of total RNA and a lower parasitemia limit of 0.05%. Finally, we designed a modified method for depletion of unwanted human hemoglobin transcripts using in vitro CRISPR-Cas9 treatment, thus improving parasite transcriptome coverage in low parasitemia samples. To prove the functionality of the pipeline for both laboratory and field strains, we generated the highest 2-hour resolution RNA-seq transcriptome for Plasmodium falciparum 3D7 intraerythrocytic lifecycle available up-to-date and also applied the entire protocol to create the largest transcriptome data from Southeast Asian field isolates.ConclusionsOverall, our methodology presents an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitemia 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.

show abstract

Post-Genomic Approaches to Understanding Malaria Parasite Biology: Linking Genes to Biological Functions

Cited by 24 publications

References 111 publications

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease

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

A Comprehensive RNA Handling and Transcriptomics Guide for High-Throughput Processing of Plasmodium Blood-Stage Samples.

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