Pfhrp2/3 deleted parasites with altered sequence coverage across regions of chromosomes 11, 13, and 5.

Sequence coverage heatmap of pfhrp3 deletion associated regions of chromosomes 11 (1,897,157 - 2,003,328 bp), 13 (2,769,916 - 2,844,777 bp), and 5 (944,389 - 988,747 bp) in the the 172 samples out of the 19,289 samples with evidence of pfhrp3 deletion. The regions from chromosomes 11 and 13 are to the end of their core region, while the region from chromosome 5 is the region around pfmdr1 that is involved in its duplication event. Each row is a WGS sample and each colun is normalized coverage. The top annotation along chromosomes depicts the location of genes with relevant genes colored: rRNA (pink), pf332 (red-orange), pfhrp3 (purple), pfmdr1 (electric-blue) and all other genes are colored light-blue. The second row delineates significant genomic regions: The chromosome 11/13 duplicated region (dark blue), the subtelomere regions of chr11/13 (orange), and the chromosome 5 duplicated region (fuchsia). The left annotation for samples includes the genomic rearrangement/deletion pattern (patterns with -TARE1 have evidence of TARE1 addition following deletion), the continent of origin, and pfhrp2/3 deletion calls. Increased variation and biases in coverage correlate with P. falciparum selective whole-genome amplification (sWGA), which adds variance and biases to the sequence coverage prior to sequencing.

Characterization of the 15.2 kb segmental duplication containing ribosomal genes on chromosomes 11 and 13.

(a) Alignment of 3D7 reference genome copies on chromosome 11 (1,918,028-1,933,288 bp) and chromosome 13 (2,792,021-2,807,295 bp). These two regions are 99.3% identical. The diagonal black bars show 100% conserved regions of at least 30 bp in length representing 89.1% of the alignment. Gene annotation is colored. (b) Comparison by pairwise alignments of the duplicated copies from non-pfhrp3 deleted strains30 assemblies (n=10) does not show a discrete separation of the paralogs with copies intermixed (chromosome 11 in blue and 13 in red). All copies are ≥99.0% similar to each other with no clear separation by continent or chromosome.

Comparison of long-read assemblies of chromosomes 11 and 13 of HB3 and SD01 to the reference genome 3D7 confirms hybridized chromosome 13-11.

Chromosome 11 of HB3 and SD01, on top, mapped entirely to the reference chromosome 11 of 3D7 with the segmental duplication region in dark blue mapped to both 11 and 13. The assembly of chromosome 13 of HB3 and SD01 maps to the reference chromosome 13 of 3D7 up through the segmental duplicated region, but after the duplication (where pfhrp3 (green) should be found), it maps to chromosome 11 of 3D7 instead of chromosome 13. Red blocks mark telomere associated repetitive elements (TARE) repeats. Displaying only from 50kb upstream from the duplicated region to the end of the chromosomes. Chromosome 11 on 3D7 spans 1,918,029 - 2,038,340 (120,311bp in length) and chromosome 13 on 3D7 spans 2,792,022 - 2,925,236 (133,214bp in length).

Long reads spanning the 15kb duplicated region confirm presence of translocated chromosome 13-11 in pfhrp3-deleted HB3 (Americas) and SD01 (Africa) but not pfhrp3-intact chromosomes.

PacBio and Nanopore reads >15kb for HB3, SD01, and CD01 are shown aligned to normal chromosomes 11 and 13 as well as hybrid chromosomes 11-13 and 13-11 constructed from 3D7 sequence. Reads that completely span the segmental duplication (dark blue) anchoring in the unique flanking sequence are shown in maroon. SD01 and HB3 only have reads that span the duplicated region on chromosome 11 but no reads that span out of the duplicated region on chromosome 13. Instead, SD01 and HB3 have spanning reads across the hybrid chromosome 13-11. Other non-deleted isolates had spanning reads mapped solely to normal chromosomes exemplified by CD01 (top row). No isolates had spanning reads across the hybrid 11-13 chromosome.

Microhaplotype patterns for the duplicated portion of chromosome 11 in 13-11++ parasites form 11 distinct haplotype groups with a geographic distinction between Africa and the Americas.

Each row represents a group of 13-11++ parasites based on shared haplotypes on the chromosome 11 duplicated segment. The number of parasites and continent of origin are on the left for each group. Each column is a different genomic region across the duplicated portion of chromosome 11. In each column, the microhaplotype is colored by the prevalence of each microhaplotype, 1=red being most prevalent, 2=orange second most prevalent, and so forth. If more than one microhaplotype for a parasite is present at a genomic location its height is relative to within-parasite frequency. Only sites with microhaplotype variation are shown (n=202). The majority of parasites show singular haplotypes at variant positions despite two copies consistent with identical haplotypes in the group and when there are multiple microhaplotypes the relative frequencies are 50/50 consistent with two divergent copies. Overall haplotype groups are markedly different, which is consistent with separate translocations emerging and spreading independently.

Proposed model of duplication-mediated non-allelic homologous recombination during intrastrain meiotic recombination yielding 13-11++ parasites.

Homology misalignment and NAHR between chromosomes 11 and 13 first occur in an oocyst formed from identical parasite gametes (intrastrain), which can then segregate, resulting in potential progeny (normal and 3 translocated progeny). Bold lines show the most direct path to a 13-11++ parasite containing a 13-11 hybrid chromosome lacking pfhrp3 and two identical copies of duplicated chromosome 11 segment seen predominantly. Subsequent recombination with an unrelated strain yields parasites with differing chromosome 11 duplication haplotypes but this can occur with subsequent interstrain meioses. Additionally, there is potential for balanced products, occurring with subsequent recombination events leading to pfhrp3 loss and either identical haplotypes (intrastrain) or different haplotypes (unrelated strain). The 11-13++ coverage pattern consistent with an 11-13 hybrid chromosome was observed once in a lab strain (FCR3), confirming it can form and thereby supporting that in vivo selective constraints prevent its emergence. Figure created using Biorender.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Genome coverage of isolates with evidence of pfhrp2 deletion.

Sequence coverage heatmap of chromosomes 8 (1,290,365 - 1,387,982 bp), 11 (1,897,157 - 2,003,328 bp), 13 (2,769,916 - 2,844,777 bp). Displaying the 27 parasites out of the 19,289 total samples that have signs of possible pfhrp2 deletions. Each row is a parasite. The top annotation along chromosomes depicts the location of genes, and the second row delineates the duplicated region (dark blue) and subtelomere region (orange). The left parasite annotation includes the deletion pattern, continent of origin, and pfhrp2/3 deletion calls. The 21 parasites that also have evidence of HRP3 deletion were only found within South America and Africa and had evidence of the 13-11++ deletion HRP3 deletion pattern. Of the 6 parasites without HRP3 deletion, 2 were from South America, 3 from Asia, and 1 from Oceania.

Coverage of sub-telomeric region of chromosome 8 before pfhrp2 of parasites with pfhrp2 deletion.

Heatmap coverage normalized to genomic coverage of the sub-telomeric region of chromosome 8 (spanning 1,365,360-1,375,435 bp, 10,075bp in length) for the 27 parasites with pfhrp2 genomic deletion. Each row is a parasite, and each column is a genomic location. The top annotates which gene the region falls within. The right side annotation shows the country of origin and which parasites have evidence of TARE1 at the location where genomic coverage drops to zero within this region. The majority of parasites without evidence of TARE1 or other genomic rearrangement are sWGA parasites and may lack the coverage to detect such events.

Coverage of chromosome 13 for parasites with pfhrp3 deletion pattern 13-.

Heatmap coverage normalized to genomic coverage of the sub-telomeric region of chromosome 13 (spanning 2,817,793 - 2,844,785 bp, 26,992bp in length) for the 50 parasites with pfhrp3 deletion pattern 13-. Each row is a parasite, and each column is a genomic location. The top annotates which gene the region falls within. The right side annotation shows which parasites have evidence of TARE1 at the location where genomic coverage drops to zero within this region (n=20) and which parasites have evidence of genomic rearrangement with chromosome 5 from discordant paired-end reads, which results in duplication of pfmdr1 (n=28). The top 2 parasites lack evidence of either deletion type. The next 28 parasites have evidence of rearrangement with chromosome 5 with discordant reads with mates mapping to chromosome 13 and other mates mapping to chromosome 5. The next 20 parasites have evidence of TARE1 contiguous with chromosome 13 sequence on various locations consistent with breakage and telomere healing. These breaks occur on chromosome 13 at 2,836,793 (n=9), 2,830,793 (n=4), 2,829,793 (n=2), 2,821,793 (n=1), 2,822,793 (n=1), 2,833,793 (n=1), 2,834,793 (n=1), 2,836,793 (n=1), and 2,840,793 (n=1).

Coverage of chromosome 5 for parasites with pfhrp3 deletion pattern 13- and 13-5++.

Heatmap coverage normalized to genomic coverage of a region of chromosome 5 (spanning 944,534 - 988,747 bp, 44,213bp in length) for parasites with pfhrp3 deletion pattern 13-. Each row is a parasite, and each column is a genomic location. The top annotates which gene the region falls within. The right side annotates the country of origin of the parasite. The 3rd annotation bar on the right side shows which parasites have TARE1 detected on chromosome 13. The parasites with green have TARE1 detected on chromosome 13 that are clustered on the bottom of the graph and have normal coverage across this region of chromosome 5, while the parasites on top have evidence of re-arrangement between chromosome 13 (position 2,835,587) and chromosome 5 (position 979,203) and show increased coverage across chromosome 5 up to the point where TARE1 sequence is detected on the reverse strand. The second annotation pink bar from the top indicates the duplicated region. The beginning of this bar is where the TARE1 sequence on the reverse strand is detected. The above would be consistent with a genomic rearrangement between chromosome 13 at position 2,835,587 and chromosome 5 at position 979,203, which results in the deletion of chromosome 13 from 2,835,587 onwards and the duplication of a 26KB region of the reverse strand of chromosome 5 from position 979,203 to 952,668 resulting in the duplication of pfmdr1 and the deletion of pfhrp3. The parasites that had transposition evidence between 13 and 5 but no TARE1 detected on 5 were all sWGA parasites, which likely limited the ability to detect the TARE1 sequence. The top 4 parasites appear coverage-wise to have 3 copies of pfmdr1. The 4 top parasites, in addition to the likely copy on chromosome 13, has a tandem duplication on chromosome 5. There were 2 different tandem duplications detected. The 1st parasite is tandemly duplicated from a monomeric stretch of Ax19 at 947,967 and Ax18 at 970,043 and is from Laos. The 2nd, 3rd, and 4th parasites, all from Cambodia, are tandemly duplicated from Ax22 at chromosome 5 946,684 and Ax36 at 964,504. All parasites with evidence for 13-5++ have only wild-type pfmdr1 or are a mix of wild-type and 184F. The four parasites with three copies of pfmdr1 have 2x coverage of 184F and 1x coverage of wild-type pfmdr1, which is consistent with the duplicated pfmdr1 on chromosome 13 being wild-type pfmdr1. All parasites with this deletion pattern are found within Asia. Of note, parasite PV0257-C, 6th from the bottom, has a COI of 2 and 2 different copies of MDR1 but normal genomic coverage.

Chromosome 5 duplicated region microhaplotypes.

The 26 microhaplotype regions across the duplicated portion of chromosome 5 952,668 to 979,203 for the 46 isolates with chromosome 13 deletion without chromosome 11 duplication. Each row is an isolate. In each column, the isolate is typed by microhaplotype (colored by the prevalence of each microhaplotype 1=red being most prevalent, 2=yellow second most prevalent, 3=purple least prevalent). This color coding system is specific to each column, and the same color across columns does not indicate the same haplotype, just the prevalence in the population for that column. Associated metadata for each isolate can be seen on the left after the isolate’s name. The majority (n=28) of these show evidence of a complex recombination with chromosome 5 at 952,668 and 2,835,587 on chromosome 13, which results in deletion of pfhrp3 and duplication of pfmdr1. Only 6 isolates with MDR duplication have no variation within pfmdr1. The other isolates have a wild type Y184 (yellow) on one copy and 184F (red) on the other copy.

Jaccard similarity between parasites for chromosome 11 duplicated segment for pfhrp3 deletion pattern 13-11++ parasites.

An all-by-all distance matrix showing Jaccard similarity for the duplicated chromosome 11 segments between all parasites with pfhrp3 deletion pattern 13-11++. The parasites’ continent, region, and country are annotated on the sides of the heatmap as well as the pfhrp2/3 deletion calls and whether the chromosome 11 duplicated segment shows an identical haplotype across the chromosome 11 duplicated segment. There are clearly several different haplotypes within the duplicated chromosome 11 segment, and there does not appear to be one specific haplotype associated with the duplication. Parasites group strongly by geographical location.

Jaccard similarity for chromosome 11 duplicated segment

All parasites with micohaplotypes similar to the duplicated chromosome 11 microhaplotypes for the pfhrp3 deletion Pattern 13-11++ parasites. While similar to Supplemental Figure 6, this all-by-all heatmap of Jaccard similarity includes all parasites with a similar chromosome segment to the parasites with pattern 13-11++ pfhrp3 deletions. For the side and top annotation for the parasites that do not have chromosome 11 duplication, there is a gray bar for whether or not they have perfect chromosome 11 duplication. There are many parasites with closely related chromosome 11 segments to the duplicated chromosome 11 segments, indicating that the duplicated chromosome 11 segments are also circulating within the population in strains with normal chromosome 11 and 13 arrangements.

Chromosome 11 Duplicated Segment pfhrp3 deletion Pattern 13-11++ parasites.

Plotted haplotype variation per sub-genomic regions across the duplicated chromosome 11 segment for the pfhrp3 pattern 13-11++ parasites. Across the x-axis are the genomic regions in genomic order, and the genomic region genes are colored on the bottom bar. Y-axis is each parasite with pattern 13-11++ of pfhrp3 deletion where this segment of chromosome 11 is duplicated onto chromosome 13. The continent, region, and country are colored per parasite on the leftmost of the plot. Each column contains the haplotypes for that genomic region colored by the haplotype rank at that window. If the column is black, there is no variation at that genomic window. Colors are done by the frequency rank of the haplotypes, and shared colors between columns do not mean they are the same haplotype. If there is more than one variant for a parasite at a genomic location, the bar’s height is the relative within-parasite frequency of that haplotype for that parasite. The parasites are ordered in the same order as the heatmap dendrogram seen in Supplemental Figure 6. There are clear distinctive haplotypes for this duplicated region.

Chromosome 11 Duplicated Segment pfhrp3 deletion Pattern 13-11++ parasites with perfect copies.

Subset of the parasites from Supplemental Figure 8 for the parasites that have a perfect duplication of the chromosome 11 segment. There are clearly very divergent haplotypes for the perfect duplications, which would indicate that the duplication event is happening multiple times and is not stemming from a single event that all parasites are descended from.

Chromosome 11 Duplicated Segment pfhrp3 deletion Pattern 13-11++ parasites with divergent chromosome 11 copies.

Subset of the parasites from Supplemental Figure 8 for the parasites that have divergent duplicates of the chromosome 11 segment. There are several parasites that have divergent chromosome 11 segments, but they share the same exact divergent copies with other parasites, which would be consistent with the coinheritance of the two divergent copies simultaneously. This could be consistent with parasites inheriting from previous duplication events involving divergent copies or meiotic recombination between parasites with two separate duplication events of disparate chromosome 11 segments, inheriting one chromosome 11 segment on chromosome 13 from parent 1 and a different chromosome 11 segment on chromosome 11 from parent 2.

Chromosome 11 Duplicated Segment coverage for pfhrp3 deletion Pattern 13-11++ parasites SD01, HB3, and Salvador 1.

Subset of the parasites from Supplemental Figure 8 but for SD01 and HB3, which were sequenced in this paper, and for Santa-Luca-Salvador-1, another lab isolate that shows similar pfhrp3 deletion pattern 13-11++. SD01 and Santa-Luca-Salvador-1 have perfect copies, while HB3 has divergent copies.

Chromosome 11/13 15.2kb duplicated region for pfhrp3 deletion pattern 13-11++ parasites.

An all-by-all distance matrix showing Jaccard similarity for the chromosome 11 and 13 duplicated region between all the parasites with pfhrp3 deletion pattern 13-11++. The top triangle is identical to the bottom triangle. Parasites’ continent, region, and country are annotated on the sides of the heatmap as well as the pfhrp2/3 deletion calls and whether the chromosome 11 duplicated segment is a perfect copy or not. Sequences tend to cluster per geographic region with similar sequences being from the same country though parasites are not as strong separately by continent as they were for the duplicated chromosome 11 segment. Despite all parasites having duplicated chromosome 11 via this region, there are clear different haplotype groups, which is consistent with multiple different origins of this duplication event.

Chromosome 11/13 15.2kb duplicated region for pfhrp3 deletion pattern 13-11++ parasites.

Plotted microhaplotype variation per subgenomic regions across the region shared between all chromosomes 11 and 13,. Across the x-axis are the genomic regions in genomic order, and the genomic region genes are colored on the bottom bar. Y-axis is each parasite with pattern 13-11++ of pfhrp3 deletion where this segment of chromosome 11 is duplicated onto chromosome 13. The continent, region, and country are colored per parasite on the left most of the plot. Each column contains the microhaplotypes for that genomic region colored by the microhaplotype rank at that window. If a column is black, there is no variation at that genomic window. Colors are by the frequency rank of the microhaplotypes, and shared colors between columns do not mean they are the same microhaplotype. If there is more than one variant for a parasite at a genomic location, the bar’s height is the relative within-parasite frequency of that microhaplotype for that parasite. Pattern 13-11++ is missing 46,323 bases from chromosome 13 (2,807,159 to 2,853,482) with a gain of 70,175 bases of chromosome 11 (1,933,138 to 2,003,313). Based on genomes that are assembled to the end of their telomeres30, an additional 17-84kb is deleted from the paralogous sub-telomeric region on chromosome 13, and an additional 15-87kb of the paralogous sub-telomeric region on chromosome 11 is duplicated.

Chromosome 11/13 15.2kb duplicated region for pfhrp3 deletion pattern 13-11++ parasites with identical chromosome 11 segment haplotypes.

Subset of parasites from Supplemental Figure 13 for the chromosome 11/13 duplicated region for the parasites with identical chromosome 11 segments based on their microhaplotypes. The left most column contains the groupings based on the microhaplotypes on chromosome 11. There are several parasites with divergent copies of the 15.2kb duplicated region despite the downstream chromosome 11 segments being a perfect copy. This would be consistent with the breakpoint for the duplication event being within this region itself where recombination occurred between nonidentical copies.

Gene Annotations of Chromosome 8 of PacBio-assembled P. Laverania Genomes.

Plots of the peri-telomere regions of chromosome 8 across all sequenced Laverania Genomes36. Assembly of this region is incomplete for the majority of strains, and only Pf3d7 and the closest relative to falciparum, PPRFG01, contain hrp2, but they are in similar locations.

Gene Annotations of Chromosome 11 of PacBio-assembled P. Laverania Genomes

Plots of the peri-telomere regions of chromosome 11 across all sequenced Laverania Genomes36. Assembly of this region is incomplete for the majority of strains. Plots begin 25kb before the rRNA loci on this region where the duplicated region between chromosomes 11 and 13 is. All strains assemblies that contain this region have this region shared between species and between chromosomes 11 and 13.

Gene Annotations of Chromosome 13 of PacBio-assembled P. Laverania Genomes

Plots of the peri-telomere regions of chromosome 13 across all sequenced Laverania Genomes36. Assembly of this region is incomplete for the majority of strains. Plots begin 25kb before the rRNA loci on this region where the duplicated region between chromosomes 11 and 13 is. All strains assemblies that contain this region have this region shared between species and between chromosomes 11 and 13.

Gene Annotations of Chromosome 11 of PacBio-assembled Genomes.

The genomic annotations across the 3′ telomeric regions of PacBio-assembled genomes30 across chromosome 11 with the telomere repetitive elements (TAREs) are also shown if present. The presence of TAREs suggests that the assembly has made its way to the sub-telomeric region (end) of the chromosome. The previously published PacBio assembled genomes for SD01 and HB3 did not reach the TAREs for chromosome 11 and terminated in the segmental duplication. The absence of an assembled sub-telomeric region on chromosome 11 prevents detailed analysis of the mechanism behind the deletion of pfhrp3 and is likely a result of the inability of the assembler and/or underlying PacBio reads to unambiguously traverse the segmental duplication and separate the duplicated chromosome 11 subtelomeric region sequence into two copies.

Gene Annotations of Chromosome 13 of PacBio-assembled Genomes.

The genomic annotations across the 3′ telomeric regions of PacBio-assembled genomes30 across chromosome 13 with the telomere repetitive elements (TAREs) are also shown if present. The presence of TAREs would suggest that the assembly has made its way all the way through the sub-telomeric region for the chromosome. The previously published PacBio-assembled genomes for SD01 and HB3 have sub-telomeric chromosome 11 sequences beginning after the segmental duplication, which is suggestive of a translocation but given the incompleteness of the chromosome 11 assembly in Supplemental Figure 18 it cannot be determined if this is simply a misassembly or a true translocation.

Chromosome 11/13 15.2kb duplicated region for parasites SD01, HB3, and Salvador 1.

Subset of the parasites from Supplemental Figure 13 but for SD01 and HB3, which were sequenced in this paper, and for Santa-Luca-Salvador-1, another lab isolate that shows similar pfhrp3 deletion pattern 13-11++. SD01 and Santa-Luca-Salvador-1 have perfect copies, but Santa-Luca-Salvador-1 has variation at 7 loci within the duplicated region, and SD01 has variation at 16 loci within this region. HB3 has divergent copies of the duplicated chromosome 11 segment and also contains variation within this region.

Spanning PacBio and Nanopore Reads across the duplicated region for SD01.

The spanning Nanopore and PacBio reads across the chromosome 11 and 13 duplicated regions for isolate SD01. The visualization truncates the reads if they span outside of the range shown. The left panel is chromosome 11, and the right panel is the hybrid chromosome 13-11. The chr11/13 duplicated region is colored in dark blue on the bottom of the plot, and the 4 loci where the isolate SD01 has key variation within this region, which can be used to optimize bridging across this duplicated region are colored pink. The reads are colored by the chromosome associated with the variation seen in each read. The association was made by linking the variation found within each of the 4 loci and looking at the reads spanning from each chromosome to see which variants were associated with which chromosome. Each locus had 2 variants and had a strong association with each chromosome.

Exact Matches between Nanopore-assembled HB3 chromosome 13 with HB3 chromosome 11, 3D7 chromosomes 11, 13.

The locations of exact matches between the Nanopore-assembled HB3 chromosome 13 and between the assembled chromosome 11 as well as the chromosomes of 3D7 11 and 13. The dark blue shaded region shows the location of the duplicated region between chromosomes 11 and 13. The assembled chromosome 13 matches the 3D7 chromosome 13 until this duplicated region and then more closely matches 3D7 chromosome 11 as well as its own chromosome 11. The figure begins 50,000bp before duplicated region, but the new HB3 chromosome 11 matches 3D7 chromosome 11 for the rest of the beginning of the contig.

Annotation of HB3 chromosomes 11 and 13-11.

The new Nanopore assembly of HB3 was annotated by Companion37, and the ends of chromosomes 11 and 13 are shown above. The duplicated region between chromosomes 11 and 13-11 is shown in blue under each chromosome, and the areas where HB3 chromosomes 11 and 13 have exact matches of at least 31bp are labeled in red underneath. Exact matches of at least 31bp to 3D7 chromosome 11 are shown in green. Both chromosomes end with telomere-associated repetitive elements (TARE), and both end with TARE1, which indicates that both assembled chromosomes reached the end of the telomere.

Annotation of SD01 chromosomes 11 and 13.

The Nanopore assembly of SD01 was annotated by Companion37, and the ends of chromosomes 11 and 13 are shown above. The duplicated region between chromosomes 11 and 13 is shown in green, and the areas where SD01 chromosomes 11 and 13 have exact matches of at least 31bp are marked out in red underneath. Exact matches of at least 31bp to 3D7 chromosome 11 are shown in green. Due to the low quality of the input DNA of the SD01 parasite, the assembly of these chromosomes did not reach the end of the telomere given the fact that these assembled contigs did not contain TARE. The assembly of these two chromosomes shows a high degree of similarity from the duplicated region to the end of the 13 associated contig (98.4% similarity with only 1,428 difference over the 89,733 base region).

Windows of interest chromosomes 8, 11, 13. The chromosomes are mapped from the beginning of the regions of interest to the chromosomes’ ends with all genes/pseudogenes annotations shown colored on top of the gray bars representing the chromosomes. From top to bottom, the regions are from chromosomes 8 (1290239-1387982), 11 (1897151-2003328), 13 (2769916-2844785). The black bars on the bottom half of each chromosome are non-paralogous regions present in all strains, as described in the Methods section. The last black bar to the end of the gray bar represent the sub-telomeric regions. The orange bars on top of the black bars are sub-regions where there is variation that can be used to type the chromosomes. The duplicated region between chromosomes 11 and 13 is shown (dark blue bars below chromosomes 11 and 13) as are the regions containing the pfhrp genes (lighter blue bars below chromosomes 11 and 13). The yellow and pink bars on the bottom of the chromosomes represent the telomere-associated tandem repeats found at the end of chromosomes.

Windows of interest chromosome 05 around pfmdr1 The windows used to investigate the duplication around pfmdr1 on chromosome 5 associated with the deletion of pfhrp3. All genes/pseudogenes annotations are shown on top of the gray bars representing the chromosome region investigated (929384-988747). The black bars on the bottom half of each chromosome are non-paralogous regions present in all strains, as described in the Methods section. The orange bars on top of the black bars are sub-regions where there is variation that can be used to type the chromosome. The pink bar shows the region that is duplicated in pattern 5++13-.

Genome coverage chromosome 8, 11, and 13 regions of isolates with subtelomere deletion of chromosome 11.

Sequence coverage heatmap showing involved regions of chromosomes 11 (1,897,157 - 2,003,328 bp), 13 (2,769,916 - 2,844,777 bp), and 5 (944,389 - 988,747 bp) in the subset of the 19,289 parasites along with key lab isolates showing evidence of deletion of putative chromosome 11 subtelomere deletions. There are 42 parasites with evidence of pfhrp3 deletions. Each row is a parasite. The top annotation along chromosomes depicts the location of genes, and the second row delineates the duplicated region (dark blue) and subtelomere region (orange). The left parasite annotation includes the deletion pattern, continent of origin, and pfhrp2/3 deletion calls. There were 42 parasites with evidence of sub-telomeric chromosome 11 deletions, 39 of which contained TARE1 sequence where coverage drops to zero, which would be consistent with telomere healing. Only one parasite (lab isolate FCR3) had deletion up and through pf332 to the ribosomal duplicated region with subsequent duplication of chromosome 13 that would be consistent with the reciprocal of 13-11++. No field parasites had this pattern. The related clone of FCR3, IT, did not contain this pattern and would suggest that FCR3 duplicated this segment of chromosome 13 via translocation within culture and not in the field.