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Single-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes. Supplementary Information The online version contains supplementary material available at 10.1186/s13073-021-00889-9.

Extrachromosomal (ec) DNAs are genetic elements that exist separately from the genome. Since ecDNA can carry beneficial genes, they are a powerful adaptive mechanism in cancers and many pathogens. For the first time, we report ecDNA contributing to antimalarial resistance in Plasmodium falciparum, the most virulent human malaria parasite. Using pulse field gel electrophoresis combined with PCR‐based copy number analysis, we detected two ecDNA elements that differ in migration and structure. Entrapment in the electrophoresis well and low susceptibility to exonucleases revealed that the biologically relevant ecDNA element is large and complex in structure. Using deep sequencing, we show that ecDNA originates from the chromosome and expansion of an ecDNA‐specific sequence may improve its segregation or expression. We speculate that ecDNA is maintained using established mechanisms due to shared characteristics with the mitochondrial genome. Implications of ecDNA discovery in this organism are wide‐reaching due to the potential for new strategies to target resistance development., The most lethal malaria parasite, Plasmodium falciparum, rapidly adapts to acquire drug resistance. For the first time, we report that highly resistant Plasmodium parasites carry additional copies of resistance‐conferring genes separate from the genome, which are known as extra‐chromosomal DNA (ecDNA). This finding provides a new model to study a fundamental mechanism of adaptation that is relevant in many microbes and may lead to a new way to target malaria.

Plasmodium vivax is increasingly the dominant species of malaria in the Greater Mekong Subregion (GMS), which is pursuing regional malaria elimination. P. vivax lineages in the GMS are poorly characterized. Currently, P. vivax reference genomes are scarce due to difficulties in culturing the parasite and lack of high-quality samples. In addition, P. vivax is incredibly diverse, necessitating the procurement of reference genomes from different geographical regions. Here we present four new P. vivax draft genomes assembled de novo from clinical samples collected in the China-Myanmar border area. We demonstrate comparable length and content to existing genomes, with the majority of structural variation occurring around subtelomeric regions and exported proteins, which we corroborated with detection of copy number variations in these regions. We predicted peptides from all PIR gene subfamilies, except for PIR D. We confirmed that proteins classically labeled as PIR D family members are not identifiable by PIR motifs, and actually bear stronger resemblance to DUF (domain of unknown function) family DUF3671, potentially pointing to a new, closely related gene family. Further, phylogenetic analyses of MSP7 genes showed high variability within the MSP7-B family compared to MSP7-A and -C families, and the result was comparable to that from whole genome analyses. The new genome assemblies serve as a resource for studying P. vivax within the GMS.

Single cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed base content. Through optimization of a quasi-linear genome amplification method, we achieve better targeting of the parasite genome over contaminants and generate coverage levels that allow detection of relatively small copy number variations on a single cell level. These improvements are important for expanding accessibility of single cell approaches to new organisms and for improving the study of adaptive mechanisms.

Antimalarial resistance is a major obstacle in the eradication of the human malaria parasite, Plasmodium falciparum. Genome amplifications, a type of DNA copy number variation (CNV), facilitate overexpression of drug targets and contribute to parasite survival. Long monomeric A/T tracks are found at the breakpoints of many Plasmodium resistance-conferring CNVs. We hypothesize that other proximal sequence features, such as DNA hairpins, act with A/T tracks to trigger CNV formation. By adapting a sequence analysis pipeline to investigate previously reported CNVs, we identified breakpoints in 35 parasite clones with near single base-pair resolution. Using parental genome sequence, we predicted the formation of stable hairpins within close proximity to all future breakpoint locations. Especially stable hairpins were predicted to form near five shared breakpoints, establishing that the initiating event could have occurred at these sites. Further in-depth analyses defined characteristics of these ‘trigger sites’ across the genome and detected signatures of error-prone repair pathways at the breakpoints. We propose that these two genomic signals form the initial lesion (hairpins) and facilitate microhomology-mediated repair (A/T tracks) that lead to CNV formation across this highly repetitive genome. Targeting these repair pathways in P. falciparum may be used to block adaptation to antimalarial drugs.

Cellular signalling is a dynamic process that underlies all aspects of organismal development, function and disease. While signal transduction at the plasma membrane has received a great deal of attention, we now appreciate that activated receptors reside at the plasma membrane for only a small fraction of their life-time. Upon activation by ligand, these receptors are often internalised and sorted into different membrane-bound compartments that allows for enhanced, modified or quenched signalling. Herein we focus on post-endocytic signalling cargo as it traverses compartments including early endosomes, multivesicular bodies, lysosomes, synaptic vesicles or exosomes. Key Concepts Much of cellular signal transduction emanates from endosomal membranes Sorting, trafficking and maturation in endosomes are major regulators of signal transduction. Exosome biogenesis and secretion is an emergent property of cell–cell communication and a target for dysregulation in disease Keywords: endosomes; exosomes; early endosomes; multivesicular bodies; phagosomes; synaptic vesicle; signal transduction; cell–cell communication