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.

24Single cell genomics is a rapidly advancing field; however, most techniques are designed for 25 mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, 26 Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed 27 base content. Through optimization of a quasi-linear genome amplification method, we achieve 28 more even genome coverage and better targeting of the parasite genome over contaminants. 29 These improvements are particularly important for expanding the accessibility of single cell 30 approaches to new organisms and cell types and for improving the study of genetic mechanisms 31 of adaptation. 32 33 Keywords: whole-genome amplification, AT-skewed genome, malaria, single cell 34 sequencing, MALBAC 35 36 Background 37 Malaria is a life-threatening disease caused by protozoan Plasmodium parasites. P. falciparum 38 causes the greatest number of human malaria deaths [1]. The clinical symptoms of malaria occur 39 when parasites invade human erythrocytes and undergo rounds of asexual reproduction by 40 maturing from early forms into late stage parasites and bursting from erythrocytes to begin the 41 cycle again [2]. In this asexual cycle, parasites possess a single haploid genome during the early 42 stages; rapid genome replication in the later stages leads to an average of 16 genome copies [2].43 44Due to a lack of an effective vaccine, antimalarial drugs are required to treat malaria. However, 45 drug efficacy is threatened by the frequent emergence of resistant populations [3]. Copy number variations (CNVs), or the amplification and deletion of a genomic element, is one of the major 47 sources of genomic variation in P. falciparum that contribute to antimalarial resistance [4][5][6][7][8][9][10][11][12][13][14][15]. 48Similar to bacteria and viruses [16][17][18], a high rate of CNVs may initiate genomic changes that 49 contribute to the rapid adaptation of this organism [7,19]. Despite the importance of CNVs, their 50 dynamics in evolving populations are not well understood. 52The majority of CNVs in P. falciparum have been identified by analyzing bulk DNA in which 53 the CNVs are present in a substantial fraction of individual parasites in the population due to 54 positive selection [8,10,11,20,21]. However, many CNVs likely remain undetected because 55 they are presumably either deleterious or offer no advantages for parasite growth or transmission 56 [22] and are therefore present in low frequency [20,22]. Currently, most CNVs are identified 57 using read-depth analysis of short read sequencing data, which derives an average signal across 58 the population. For this reason, genetic variants must be present in a high frequency (i.e. ~50%) 59 in the population to be detected [23][24][25]. Sequencing at very high depth improves the detection 60 of low frequency CNVs, but the sensitivity is limited to large-scale CNVs present in > 5% cells 61 [26][27][28]. Analysis methods that rely on the detection of reads that span CNV junctions h...

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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. 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.