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opencc-zeroJan 2024View details →

A pan-cetacean MHC amplicon sequencing panel developed and evaluated in combination with genome assemblies

The major histocompatibility complex (MHC) is a highly polymorphic gene family that is crucial in immunity, and its diversity can be effectively used as a fitness marker for populations. Despite this, MHC remains poorly characterised in non-model species (e.g., cetaceans: whales, dolphins and porpoises) as high gene copy number variation, especially in the fast-evolving class I region, makes analyses of genomic sequences difficult. To date, only small sections of class I and IIa genes have been used to assess functional diversity in cetacean populations. Here, we undertook a systematic characterisation of the MHC class I and IIa regions in available cetacean genomes. We extracted full-length gene sequences to design pan-cetacean primers that amplified the complete exon2 from MHC class I and IIa genes in one combined sequencing panel. We validated this panel in 19 cetacean species and described 354 alleles for both classes. Furthermore, we identified likely assembly artefacts for many MHC class I assemblies based on the presence of class I genes in the amplicon data compared to missing genes from genomes. Finally, we investigated MHC diversity using the panel in 25 humpback and 30 southern right whales, including four paternity trios for humpback whales. This revealed copy-number variable class I haplotypes in humpback whales, which is likely a common phenomenon across cetaceans. These MHC alleles will form the basis for a cetacean branch of the Immuno-Polymorphism Database (IPD-MHC), a curated resource intended to aid in the systematic compilation of MHC alleles across several species, to support conservation initiatives.

Datasets of mineral composition and geochronological result of Barrovian metamorphic sequence rocks in the Ailao Shan massif

This file includes the supporting information of the manuscript titled“Thermal and physical properties of Barrovian metamorphic sequence rocks in the Ailao Shan-Red River shear zone, and implications for crustal channel flow”

opencc-by-4.0Aug 2023View details →

Single-cell RNA sequencing reveals dysregulated cellular programs in the inflamed epithelium of Crohn's disease patients.

Crohn’s disease (CD) is a complex inflammatory disorder of incompletely understood molecular aetiology. We generated a large single-cell RNA sequencing dataset from the terminal ileal biopsies of two independent cohorts comprising a total of 50 CD patients and 71 healthy controls. We performed transcriptomic analyses to reveal genes, cell types and mechanisms perturbed in CD, leveraging the power of the two cohorts to confirm our findings and assess replicability. In addition to mapping widespread alterations in cytokine signalling, we provide evidence of pan-epithelial upregulation of MHC class I genes and pathways in CD. Using non-negative matrix factorization we revealed intra- and inter-cellular upregulation of expression programs such as G-protein coupled receptor signalling and interferon signalling, respectively, in CD. We observed an enrichment of CD heritability among marker genes for various activated T cell types and myeloid cells, supporting a causal role for these cell-types in CD aetiology. Comparisons between our discovery and replication cohort revealed significant variation in differential gene-expression replicability across cell types. B, T and myeloid cells showed particularly poor replicability, suggesting caution should be exercised when interpreting unreplicated differential gene-expression result in these cell types. Overall, our results provide a rich resource for identifying cell-type specific biomarkers of Crohn’s disease and identifying genes, cell types and pathways that are causally and replicably associated with disease.

Supplementary material Next-generation sequencing reveals that miR-16-5p, miR-19a-3p, miR-451a and miR-25-3p cargo in plasma extracellular vesicles differentiates sedentary young males from athletes.

A sedentary lifestyle is a leading risk factor for global mortality. No objective molecular biomarker of sedentarism is available. Extracellular vesicles miRNAs have been described to respond to exercise. Our aim was to identify the extracellular vesicle miRNA profile of chronically trained young male athletes, endurance and resistance, compared to their sedentary counterparts. A descriptive case-control design with 16 sedentary young men, 16 Olympic male endurance athletes and 16 Olympic male resistance athletes. Next Generation Sequencing and RT-qPCR, external and internal validation, were performed in order to analysed extracellular vesicle miRNA profiles. miR-16-5p, miR-19a-3p and miR-451a were significantly upregulated in SED compared to END and RES. Besides, miR-25-3p was specifically down-regulated in END compared to SED. Extracellular vesicle miR-16-5p, miR-19a-3p, miR-451a provide an objective signature of sedentarism irrespective of the type of exercise and miR-25-3p as a specific responder to endurance training. Therefore, this study provides for the first time an objective measure to categorise individuals as sedentary or trained in young male population and moreover, it highlights a common epigenetic modulation between models of training.

opencc-by-4.0Jan 2024View details →

Comprehensive, targeted eukaryotic metagenomics analysis of environmental DNA biodiversity using Oxford Nanopore sequencing

Metagenomics has become a prominent technology for studying the functional potential of all organisms in a microbial and eukaryotic community. The study of symbiotic organisms from different classes or kingdoms, including those previously unknown, is possible with simultaneous and equally efficient metagenomic analysis of these species. A variety of targeted primer sets are used for eukaryotic metagenomic biodiversity, including those that are universal for specific families, classes, or kingdoms. The most universal for all existing cellular organisms is the presence of ribosomal RNA encoding gene sequences. For eukaryotic sequences, these are 16S and 23s rDNA, and for eukaryotic sequences of nuclear (18S and 28S) and mitochondrial (12S and 16S) ribosomal RNA. Here we present the application of the eukaryotic metagenomics approach to the simultaneous, quantitative, and unbiased identification of most eukaryotic species. To achieve this, we have developed a universal PCR assay that targets the most conservative nuclear regions of the ribosomal gene for all cellular organisms, including plants, algae, fungi, protists, insects, and animals. The amplification product contains polymorphic regions of both ribosomal genes and the intergenic spacer. The size of the PCR products varies by class, kingdom, or domain, ranging from 2 kb for fungi to 7 kb for birds. This assay is also adapted for use with the Oxford Nanopore Rapid Barcoding Library Kit, which enables metagenomic biodiversity analysis. Our approach provides a rapid, sensitive, and equally efficient way to study the composition of eDNA from mixed species in the environment. This protocol reduces the time and cost of metagenomic biodiversity analysis using Oxford Nanopore sequencing. We can efficiently analyze the biodiversity of mixed species present in environmental samples.

.bam alignment files of Illumina and ONT sequencing of pREF plasmid

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides the first universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

16S sequences from mesocosms experiment testing the effect of Siganus rivulatus on marine microorganisms

Nutrient cycling is a key biogeochemical process underlying the functioning of marine ecosystems. Yet, the contribution of fishes is still poorly understood. This is problematic considering the current modifications of fish assemblages experienced in certain regions such as the Mediterranean Sea, and their potential consequences in terms of ecosystem functioning. In this study, we used a mesocosm experiment to test the effect of nutrient recycling by an invasive marine herbivorous fish (Siganus rivulatus) on planktonic and benthic microbial communities. The response of these communities was assessed using a variety of analytical approaches such as measures of nutrient concentration, flow cytometry, and metabarcoding of the 16S rRNA gene. Our results show that several microbial compartments of marine ecosystems respond to the nutrients released by fish through excretion and egestion. The nutrients contained in the macroalgae consumed by S. rivulatus were excreted in large amounts as dissolved nutrients, which resulted in higher concentrations of N-based nutrients in the water (NH4, NO2/NO3). This excess of N in the system was associated with higher abundances of planktonic microbes (phyto- and bacterioplankton), modifications of the structure of planktonic bacterial communities, and the tissue composition of the remaining macroalgae. Non-assimilated nutrients were released in the form of feces under the shelters where the fish spent most of their time and defecated during the night, leading to local increases in diversity and significant shifts in the structure of sediment bacterial communities. Overall, our results suggest that the impact of S. rivulatus on planktonic microbes was related to the indirect bottom-up effect induced by excreted nutrients while its effect on benthic microbes was due to a direct release of microbes from its gut microbiome. This study represents one of the first assessment of the effect of nutrient recycling by fishes on the microbial communities from several compartments of marine ecosystems and one of the first evidence of the invisible effect of invasive species on the microbial components of marine ecosystems.

16S rRNA sequences from Siganidae (S. rivulatus and S. luridus) gut microbiome in their native (Red Sea) and invaded (Mediterranean Sea) ranges

The microbiome of invasive species is increasingly seen as a potential key factor of their ecological success, and this appears particularly true in herbivorous invaders whose digestive abilities rely on the microbes hosted in their gut. We characterized the gut microbiome of two invasive herbivorous fishes (Siganus rivulatus and Siganus luridus) in their native (Red Sea) and invaded (Levantine Sea and Northern Crete) ranges. We found that gut bacterial communities contain a higher taxonomic and phylogenetic diversity while becoming increasingly different from the native microbiome as the fishes move away from the native zone. This shift resulted in the homogenization of the microbiomes between individuals from the same species as well as between the two species. Firmicutes and Tenericutes reduced drastically in abundance while Proteobacteria and Bacteroidetes became more dominant in both species. This led to a modification of the functional potential of the gut microbiome associated with the metabolism of short-chanin fatty acids that also became more homogeneous in the invaded range. Altogether, our results suggest that the plasticity of the gut microbiome in Siganidae could be a key factor underlying their ecological success in Mediterranean ecosystems.

Contrasting patterns of sequence variation in steelhead populations reflect distinct evolutionary processes

Multiple evolutionary processes influence genome‐wide allele frequencies and quantifying effects of genetic drift, and multiple forms of selection remain challenging in natural populations. Here, we investigate variation at major effect loci in contrast to patterns of neutral drift across a wide collection of steelhead (Oncorhynchus mykiss) populations that have declined in abundance due to anthropogenic impacts. Whole‐genome resequencing of 74 populations of steelhead revealed genome‐wide patterns (~8 million SNPs) consistent with expected neutral population structure. However, allelic variation at major effect loci associated with adult migration timing (chromosome 28: GREB1L/ROCK1) and age at maturity (chromosome 25: SIX6) reflected how selection has acted on phenotypic variation in contrast with neutral structure. Variation at major effect loci was influenced by evolutionary processes with differing signals between the strongly divergent Coastal and Inland lineages, while allele frequencies within and among populations within the Inland lineage have been driven by local natural selection as well as recent anthropogenic influences. Recent anthropogenic effects appeared to have influenced the frequency of major effect alleles including artificial selection for specific traits in hatchery stocks with subsequent gene flow into natural populations. Selection from environmental factors at various scales has also likely influenced variation for major effect alleles. These results reveal evolutionary mechanisms that influence allele frequencies at major effect loci that are critical for conservation of phenotypic traits and life history variation of this protected species.

Alignments of reindeer/caribou mitogenome sequences

Climate warming at the end of the last glacial period had profound effects on the distribution of cold-adapted species. As their range shifted towards northern latitudes, they were able to colonise previously glaciated areas, including remote Arctic islands. However, there is still uncertainty about their colonisation routes and timings. At the end of the last ice age, reindeer/caribou (Rangifer tarandus) expanded to the Holarctic region and colonised the archipelagos of Svalbard and Franz Josef Land. Earlier studies have proposed two possible colonisation routes, either from the Eurasian mainland or from Canada via Greenland. Here, we used 174 ancient, historical, and modern mitogenomes to reconstruct the phylogeny of reindeer across its whole range and to infer the colonisation route of the Arctic islands. Our data shows a close affinity among Svalbard, Franz Josef Land, and Novaya Zemlya reindeer. We also found tentative evidence for positive selection in the mitochondrial gene ND4, which is possibly associated with increased heat production. Our results thus support a colonisation of Arctic archipelagos from the Eurasian mainland and provide some insights into the evolutionary history and adaptation of the species to its High Arctic habitat.

Alignment of mitogenome sequences (FASTA file) for a paleogenomic investigation of overharvest implications in an endemic wild reindeer subspecies

Overharvest can severely reduce the abundance and distribution of a species and thereby impact its genetic diversity and threaten its future viability. Overharvest remains an ongoing issue for Arctic mammals, which due to climate change now also confront one of the fastest changing environments on Earth. The high-Arctic Svalbard reindeer (Rangifer tarandus platyrhynchus), endemic to Svalbard, experienced a harvest-induced demographic bottleneck that occurred during the 17–20th centuries. Here we investigate changes in genetic diversity, population structure, and gene-specific differentiation during and after this overharvesting event. Using whole-genome shotgun sequencing, we generated the first ancient and historical nuclear (n = 11) and mitochondrial (n = 18) genomes from Svalbard reindeer (up to 4000 BP) and integrated these data with a large collection of modern genome sequences (n = 90), to infer temporal changes. We show that hunting resulted in major genetic changes and restructuring in reindeer populations. Near-extirpation followed by pronounced genetic drift have altered the allele frequencies of important genes contributing to diverse biological functions. Median heterozygosity was reduced by 23%, while the mitochondrial genetic diversity was reduced only to a limited extent, likely due to already low pre-harvest diversity and a complex post-harvest recolonization process. Such genomic erosion and genetic isolation of populations due to past anthropogenic disturbance will likely play a major role in metapopulation dynamics (i.e., extirpation, recolonization) under further climate change. Our results from a high-arctic case study therefore emphasize the need to understand the long-term interplay of past, current, and future stressors in wildlife conservation.

opencc-by-4.0Aug 2019View details →

MinION sequencing of SAMD00180470

Data from <a href="https://doi.org/10.1128/MRA.01212-19">https://doi.org/10.1128/MRA.01212-19</a> via <a href="https://ddbj.nig.ac.jp/public/ddbj_database/dra/fastq/DRA008/DRA008776/DRX178031/">https://ddbj.nig.ac.jp/public/ddbj_database/dra/fastq/DRA008/DRA008776/DRX178031/</a> This is a re-issue of <a href="../records/4534098">https://zenodo.org/records/4534098</a> due to the original files consisting of multiple concatenated bzip2 files, they contain far too many `BZh9` sequences (expected in the bzip compression level 9 header), resulting in a number of tools such as FastQC and Trimmomatic failing to process them with strange errors.

opencc-zeroDec 2023View details →

Data from: Evaluating genotyping-in-thousands by sequencing as a genetic monitoring tool for a climate sentinel mammal using non-invasive and archival samples

Genetic tools for wildlife monitoring can provide valuable information on spatiotemporal population trends and connectivity, particularly in systems experiencing rapid environmental change. Though many DNA sequencing approaches still require high quality and quantity of DNA obtained from traditional sources (e.g. blood and tissue), rapid genotyping tools such as Genotyping-in-Thousands by sequencing (GT-seq) have improved our ability to make use of degraded and less concentrated DNA commonly obtained from non-invasive and archival samples. Here, we developed a multi-purpose GT-seq panel (307 single nucleotide polymorphisms) for a climate sentinel mammal (the American pika, Ochotona princeps) for use as a genetic tool for monitoring populations in the Canadian Rocky Mountains. We optimized the panel using contemporary tissue samples (n = 77) and subsequently applied it to archival tissue (n = 17) and contemporary fecal pellet samples (n = 129) to evaluate its effectiveness at identifying individuals and sex, estimating relatedness, and inferring population structure. The panel demonstrated high efficacy with contemporary and archival tissue samples (94.7% and 90.5% genotyping success, respectively) and negligible genotyping error (0.001% and 0.0%, respectively). Despite relatively high genotyping success for fecal pellet samples (79.7%), high genotyping error (28.4%) limited its power as a monitoring tool to assess genetic variation using non-invasive samples and highlighted the need for further optimization around sample and data collection.

opencc-by-4.0Jan 2024View details →

The alignments of chloroplast genome sequences and nuclear ribosomal DNA fragments of six oak species sampled in the hot-dry valley of the Jinsha River, southwestern China

Both chloroplast (cp) genome sequences and nuclear ribosomal (nr) DNA were assembled using GetOrganelle v.1.7.6.1 for 18 oak trees sampled in the Panzhihua Cycad National Nature Reserve, Sichuan Province, China. These trees belong to six oak species, including Quercus cocciferoides, Q. dolicholepis, Q. franchetii, Q. griffithii, Q. longispica, and Q. variabilis. We used PhyloSuite v.1.1.152 to extract coding sequences (CDSs), tRNA genes, rRNA genes, introns, and intergenic spacers (IGSs) of the 18 oak cp genomes. These sequences were aligned separately using MAFFT v.7.3.13 and manually adjusted with BioEdit v.7.2.5. Length variations in mononucleotide repeats were excluded and inversions were replaced with their reverse complements because of their tendency for homoplasy. Other indels were coded as binary characters according to the simple gap coding method using GapCoder. Separate assignments were concatenated according to their respective positions in the cp genome to obtain the alignments of LSC, SSC, IRb, and the whole cp genome.

opencc-zeroMar 2024View details →

Control over Conformational Landscapes of Polypeptoids by Monomer Sequence Patterning

This dataset accompanies the article "Control Over Conformational Landscapes of Polypeptoids by Monomer Sequence Patterning" by Audra J. DeStefano, Shawn D. Mengel, Morgan W. Bates, Sally Jiao, M. Scott Shell, Songi Han, and Rachel A. Segalman in Macromolecules in 2024. The article demonstrates how patterning of hydrophobic residues along a polymer backbone can tune the distribution of end-to-end distances and that these effects can be predicted by a simple bead-spring simulation. This dataset contains the necessary experimental data to reproduce the main text and supporting figures. High performance liquid chromatographs, mass spectra, double electron-electron resonance time domain signals, and simulated end-to-end distance distributions are included.

Shark-dust: Application of high-throughput DNA sequencing of processing residues for trade monitoring of threatened sharks and rays

Data repository accompanying manuscript titled of "Shark-dust: Application of high-throughput DNA sequencing of processing residues for trade monitoring of threatened sharks and rays." Prasetyo, A. P., Murray, J. M., Kurniawan, M. F. A. K., Sales, N. G., McDevitt, A. D., & Mariani, S. (2023). Shark-dust: Application of high-throughput DNA sequencing of processing residues for trade monitoring of threatened sharks and rays. Conservation Letters, 16, e12971. https://doi.org/10.1111/conl.12971

opencc-zeroMar 2024View details →

Data from: Genomic footprint of cladogenesis revealed through RADseq and Sanger sequencing demonstrates congruent patterns in the velvet worm Peripatopsis sedgwicki species complex (Onychophora: Peripatopsidae)

In the present study, first generation DNA sequencing (mitochondrial cytochrome c oxidase subunit one, COI) and reduced-representative genomic RADseq data were used to understand the patterns and processes of diversification of the velvet worm, Peripatopsis sedgwicki species complex across its distribution range in South Africa. For the RADseq data, three datasets (two primary and one supplementary) were generated corresponding to 1259 - 11,468 SNPs, in order to assess the species diversity and phylogeographic of the species complex. Tree topologies for the two primary datasets were inferred using maximum likelihood and Bayesian inferences methods. Phylogenetic analyses using the COI datasets retrieved four distinct, statistically well-supported clades within the species complex. Five species delimitation methods applied to the COI data (ASAP, bPTP, bGMYC, STACEY, and iBPP) all showed support for the distinction of the Fort Fordyce Nature Reserve specimens. In the main P. sedgwicki species complex, the species delimitation methods revealed a variable number of operational taxonomic units and overestimated the number of putative taxa. Divergence time estimates coupled with the geographic exclusivity of species and phylogeographic results suggest recent cladogenesis during the Plio/Pleistocene. The RADseq were subjected to a principal components analysis and a discriminant analysis of principal components, under a maximum-likelihood framework. The latter results corroborate the four main clades observed using the COI data, however, applying additional filtering revealed additional diversity. The high overall congruence observed between the RADseq and COI data suggests that first generation sequence data remain a cheap and effective method for evolutionary studies, although RADseq does provide a far greater resolution of contemporary temporo-spatial patterns.

A Large Fault Partially Reactivated During Two Contiguous Seismic Sequences in Central Italy: The Role of Geometrical and Frictional Heterogeneities

Moment tensor catalog for events with M > 3.0, that occurred between January 2009 and April 2021, in Campotosto area, Italy. Moments tensor were calculated by applying the Time Domain Moment Tensor technique, originally proposed by Dreger and Helmberger (1993) and Pasyanos et al. (1996) and successively implemented at INGV by Scognamiglio et al. (2009). The catalog includes: Location of events: time, depth, lat and lon The moment magnitude: Mw The double-couple value: DC The Variance Reduction value: VR The six moment tensor components: Mxx, Mxy, Mxz, Myy, Myz, Mzz The orientation of nodal planes: strike1, dip1, rake1, strike2, dip2, rake2

opencc-by-4.0Feb 2022View details →

(Extended Data) Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

These extended data accompany the manuscript: Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial Table S1: Concentration ratios and resulting parasitemia in artificial dna mixtures of P. falciparum Lab Isolates 3D7 and Dd2. This table presents the parasitemia for the artificial mixtures of P. falciparum lab isolates 3D7 and Dd2. Each mixture was prepared at varying ratios of 3D7 to Dd2, starting from equal proportions to a complete presence of only 3D7. The original concentration of each isolate was approximately 50,000 parasites per microliter (pf/μl), and the table displays the proportion of each strain in the mixture and the resulting total parasitemia concentration. Table S2. List of PCR and deep sequencing primers. This table shows the list of forward and reverse primers used for deep sequencing. In boldface are the MID tags, while in the regular face are the forward primers Table S3. The relative frequencies of each ama1 variant and the number of samples with each variant. The relative frequencies (%) of the 33 AMA1 variants in pre-and post-treatment samples (n = 330) are shown as a 33 amino acid sequence. The frequencies were calculated by dividing the number of reads of each microhaplotype by the total number of reads obtained per sample (116,187,131). Table S4. Distribution of microhaplotypes among samples. This table shows the occurrence of microhaplotypes across all participants, both with monoclonal and multiclonal ama1 infections. It presents the ama1 clonality – monoclonal or multiclonal (column 1) - participant IDs (column 2), microhaplotype IDs (column 3), and the relative frequencies of these microhaplotypes across timepoints from 0 to 1008 hours (day 42) (column 3). Dashes represent time points where microhaplotypes were missing or were not detected. Table S5. Distribution of rare microhaplotypes among samples. This table shows the occurrence of rare microhaplotypes in various samples. It presents participant IDs (column 1), microhaplotype IDs (column 2), and the relative frequencies of these microhaplotypes across time points from 0 to 1008 hours (day 42) (column 3). Samples containing rare microhaplotypes - specifically from PID10, PID32, PID38, PID40, PID49, PID60, PID63, and PID65 - are shown in orange, along with the corresponding rare microhaplotypes and their time points of occurrence. Furthermore, participants are categorised by shared microhaplotypes to indicate instances of rarity and commonality. Except for one microhaplotype unique to PID30, rare microhaplotypes were detected in several samples, frequently exceeding a 5% relative frequency. Dashes represent time points where microhaplotypes were missing or were not detected. Table S6. The parasitemia levels associated with each ama1 microhaplotype per timepoint. This table shows the parasitemia for each ama1 microhaplotype per timepoint and each participant. “Patient ID” represents the patient ID, “AMA1 COI at 0h” represents the complexity of infection (COI) for each participant at baseline, based on ama1 while subsequent columns represent the parasitemia for each ama1 microhaplotype from timepoint 0h to 1008h. Parasitemia was back-calculated using the COI and total parasitemia for each time point. For time points with a COI > 1, parasitemia for the respective ama1 microhaplotypes are separated by commas, cells in red indicate timepoints without sequencing data (ND = not determined). In contrast, cells in grey indicate time points where microhaplotypes were detected below 10 parasites/μl, hence at risk of falling below the sampling limit. Figure S1. Performance of AmpSeq in the sequencing controls. Six aliquots were prepared for each control set to ensure sufficient control data in case of PCR or sequencing failure. The median read depth in the lab controls was 5,658 (range 4,310 – 12,603) and 704 (291 – 1,676). The x-axis represents the aliquot identifier across the five mixtures, starting from 1 to 6, while the y-axis represents the proportions of each variant across all aliquots. For ama1 (A), two variants (3D7 and Dd2) were detected, whereas in mdr1 (B), two variants were detected YY, FY and NY following amplification of Dd2 Copy I, Dd2 Copy II and 3D7, respectively. For ama1, sequencing failed for aliquot 6 of control set 1, while for mdr1, sequencing failed for aliquot 2 and 6 of control set 3, aliquots 1 and 6 of control set 4 and aliquots 1 and 5 of control set 5. Under the mdr1 control set 4, the Dd2 copy II (86F, 184Y) was not identified, possibly due to having very low concentrations that were not picked up in this aliquot. Based on our control mixtures, the minimum variant frequency we could detect was 5%. Figure S2. Heatmaps of the successfully PCR amplified and sequenced samples for ama1 (A) and mdr1 (B). The rows represent the study participants, while the columns represent time in hours. Successfully sequenced samples are shown in blue, those that failed PCR are shown in red and those that failed sequencing are in black. The timepoint “ Rec” represents unscheduled visits where a recurrent sample was collected. The unshaded areas with "-" are time points where samples were not collected. For each time point, the number of samples successfully sequenced (n Successful) is indicated in the last row of each panel. The table in panel C shows the groupings of samples based on parasitemia, high (> 5,000), moderate (100-5,000) and low (< 100 parasites per microlitre). Many samples collected between 0h-12h had high parasitemia, samples collected between 18h–30h had moderate parasitemia, while samples collected after 30h were primarily of low parasitemia. Figure S3. The mean complexity of infection (COI) by AMA1 throughout treatment. The mean COI (red diamonds) appeared to be stable (between 1.5 - 2) from baseline (0h) up to 72h and thereafter fluctuated due to the small sample sizes (<5) in the post-treatment samples. The black dots represent the COI per sample.