Qiacube robot

Total RNA including miRNAs was isolated from blood samples using PAXgene Blood miRNA Kit on the QIAcube robot (Qiagen, Hilden, Germany) following the manufacturer's recommendations. DNase I treatment (Qiagen, Hilden, Germany) was carried out during the isolation to eliminate any genomic DNA contamination as previously described^{9 (link)}. The total RNA concentration was measured using the NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, United States). RNA purity was assessed by determining the OD 260/280 and the OD 260/230 ratios. The quality of total RNA was assessed using the Agilent Bioanalyser 2100 Eukaryote Total RNA Nano Series II (Agilent Technologies, California, United States).

All DBSs for the pfhrp2 and pfhrp3 studies were shipped to the Australian Defence Force Malaria and Infectious Disease Institute (ADFMIDI) where molecular testing was conducted. From each DBS sample, three discs of DBS were punched into 1.5-mL microfuge tubes. DNA was extracted using QIAamp DNA Mini Kits and a QIAcube Robot (QIAGEN, Crawley, UK) according to the manufacturer’s instructions. Samples were eluted into a volume of 100 µl with AE buffer. A P. falciparum-positive control DBS spot was extracted and processed in each run alongside samples. Details of the QIAamp DNA Mini Kits and a QIAcube Robot extraction method has been described and published elsewhere [12 (link), 17 (link), 25 (link), 36 (link), 37 (link)].

Each dried blood spot was processed into 3 discs from which genomic DNA was extracted by using QIAamp DNA Mini Kits and a QIAcube Robot (QIAGEN, Crawley, UK) according to the manufacturer’s instructions. DNA was eluted into a volume of 100 µL, and 10 µL was used in each PCR. An 18S rRNA gene-based multiplex PCR (20 (link)) was used to determine whether 4 human Plasmodium spp. were present in each sample.

Extraction of genomic DNA from blood on filter papers was conducted using QIAGEN QIAamp DNA Mini Kits and a QIAcube robot (QIAGEN, Crawley, U.K.). The manufacturer’s protocol (QIAamp DNA Mini and Blood Mini Handbook 2E) was followed, except only one 5 mm circle was extracted per sample, and eluted to a 100 μL volume. Detection for Plasmodium species was undertaken using the method described previously [23 (link)]. DNA samples that were determined positive for P. vivax were used for genotyping.

Extractions of genomic DNA were performed using the Gentra Puregene Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. Additional 15 P. nobilis individuals (Peyrefite bay, Banyuls, France) and 23 P. rudis (Cabrera, Balearic Island, Spain) were added to the study for genotype comparison and DNA was extracted using the QIACube robot (Qiagen, Hilden, Germany) according to manufacturer’s instructions.

DNA was extracted from three replicate 0.25 g soil samples from each plot using the Qiagen DNeasy Powersoil Pro Kit (Qiagen, Germantown, MD). The extraction process was carried out using a fully automated Qiagen QIAcube robot with a 10-min vortex lysis step. DNA quality was assessed using a Nanodrop 1000 (Thermo Scientific, Waltham, MA) and quantified fluorometrically with the Invitrogen dsDNA HS Assay Kit on a Qubit 2.0 (Life Technologies, Carlsbad, CA).

Total DNA was extracted from EDTA-collected peripheral blood using the MagAttract DNA blood Midi M48 Kit on a QIAcube robot (Qiagen). DNA concentrations were measured using the Quant-iT Picogreen ds DNA Reagents (Invitrogen). Individuals were genotyped for >700 K SNPs using Illumina’s Human OmniExpress BeadChips, an iScan system and the Genome Studio software following the guidelines of the manufacturer. We eliminated variants with call rate ≤0.95, deviating from Hardy–Weinberg equilibrium (p ≤ 10⁻⁴), or which were monomorphic. We confirmed European ancestry of all individuals by PCA using the HapMap population as reference. Using the real genotypes of 629,570 quality-controlled autosomal SNPs as anchors, we used the Sanger Imputation Services with the UK10K + 1000 Genomes Phase 3 Haplotype panels (https://imputation.sanger.ac.uk)^{35 –37 (link)} to impute genotypes at autosomal variants in our population. We eliminated indels, SNPs with MAF ≤ 0.05, deviating from Hardy-Weinberg equilibrium (p ≤ 10⁻³), and with low imputation quality (INFO ≤ 0.4), leaving 6,019,462 high quality SNPs for eQTL analysis.