Phi29 reaction buffer

We used Qiagen DNA mini kit to extract and purify the genomic DNA, and Quant-iT^™ PicoGreen^® Assay (Invitrogen) to quantify the amount of DNA. For samples with less than 50ng DNA obtained, whole genome amplification (WGA) was performed before NGS library preparation. WGA reactions were performed following Nair et al (Nair et al., 2014). Each 25 μl reaction contained at least 5ng of Plasmodium DNA, 1× BSA (New England Biolabs), 1 mM dNTPs (New England Biolabs), 3.5 μM of Phi29 Random Hexamer Primer, 1× Phi29 reaction buffer (New England Biolabs), and 15 units of Phi29 polymerase (New England Biolabs). We used a PCR machine (SimpliAmp, Applied Biosystems) programmed to run a “stepdown” protocol: 35 °C for 10 min, 34 °C for 10 min, 33 °C for 10 min, 32 °C for 10 min, 31 °C for 10 min, 30 °C for 6 h then heating at 65 °C for 10 min to inactivate the enzymes prior to cooling to 4 °C. Samples were cleaned with AMPure XP Beads (Beckman Coulter) at a 1:1 ratio. We constructed next generation sequencing (NGS) libraries using 50–100 ng DNA or WGA product following the KAPA HyperPlus Kit protocol with 3-cycle of PCR. All libraries were sequenced at 150bp pair-end using Illumina Novaseq S4 or Hiseq X sequencers. We sequenced all bulk samples to a minimum coverage of 100×.

The sWGA method was performed on genomic DNA from unfiltered samples according to published protocols [9 (link)]. The sWGA reaction was performed in 0.2 mL PCR-tubes, containing 10 ng of template genomic DNA, 1 × BSA (New England Biolabs), 1 mM dNTPs (New England Biolabs), 2.5 µM of each amplification primer (Additional file 1: Table S2), 1 × Phi29 reaction buffer (New England Biolabs), 30 units of Phi29 polymerase (New England Biolabs), and molecular biology grade water to reach a final reaction volume of 50 µL. The reaction was carried out on a thermocycler with the following step-down program: 5 min at 35 °C, 10 min at 34 °C, 15 min at 33 °C, 20 min at 32 °C, 30 min at 31 °C, 16 h at 30 °C, then heating for 15 min at 65 °C to inactivate the Phi29 polymerase before cooling to 4 °C. Amplified products were quantified using the Qubit® dsDNA high sensitivity kit (Thermo Fisher Scientific) to determine whether there was at least 500 ng of product for sequencing. Amplified products were cleaned using Agencourt Ampure XP beads (Beckman Coulter) as follows: 1.8 volumes of beads were added to 1 volume of amplified products, briefly mixed, and then incubated for 5 min at room temperature. A magnetic rack was used to capture the DNA binding beads. The DNA binding beads were then washed twice using 200 µL of 80% ethanol and eluted with 60 µL of EB buffer.

The sWGA reactions were performed using one set of primers for P. ovale curtisi and one set of primers for P. ovale wallikeri. Each reaction was performed in 0.2 mL PCR tubes containing at least 20 ng of template genomic DNA, 0.1 mg/mL bovine serum albumin (BSA) (New England Biolabs), 1 mM dNTPs (New England Biolabs), 2.5 μM each primer, 1× Phi29 reaction buffer (New England Biolabs), 30 units of Phi29 polymerase (New England Biolabs), and molecular biology-grade water to reach a final reaction volume of 50 μL. The reaction was carried out on a thermocycler (Mastercycler Gradient, Eppendorf) with the following step-down program: 5 min at 35°C, 10 min at 34°C, 15 min at 33°C, 20 min at 32°C, 25 min at 31°C, 16 h at 30°C, then heating for 15 min at 65°C to inactivate the Phi29 polymerase before cooling to 4°C. Amplified products were quantified using the Qubit dsDNA high-sensitivity kit (Thermo Fisher Scientific). Amplified products were cleaned using Agencourt Ampure XP beads (Beckman Coulter) as follows: 1.8 volumes of beads were added to 1 volume of amplified products, briefly mixed, and then incubated for 5 min at room temperature. A magnetic rack was used to capture the DNA binding beads. The beads were then washed twice using 200 μL of 80% ethanol, and DNA was eluted with 60 μL of EB buffer.

The sWGA reactions were performed using one set of primers for P. ovale curtisi and one set of primers for P. ovale wallikeri. Each reaction was performed in 0.2 mL PCR tubes containing at least 20 ng of template genomic DNA, 0.1 mg/mL bovine serum albumin (BSA) (New England Biolabs), 1 mM dNTPs (New England Biolabs), 2.5 μM each primer, 1× Phi29 reaction buffer (New England Biolabs), 30 units of Phi29 polymerase (New England Biolabs), and molecular biology-grade water to reach a final reaction volume of 50 μL. The reaction was carried out on a thermocycler (Mastercycler Gradient, Eppendorf) with the following step-down program: 5 min at 35°C, 10 min at 34°C, 15 min at 33°C, 20 min at 32°C, 25 min at 31°C, 16 h at 30°C, then heating for 15 min at 65°C to inactivate the Phi29 polymerase before cooling to 4°C. Amplified products were quantified using the Qubit dsDNA high-sensitivity kit (Thermo Fisher Scientific). Amplified products were cleaned using Agencourt Ampure XP beads (Beckman Coulter) as follows: 1.8 volumes of beads were added to 1 volume of amplified products, briefly mixed, and then incubated for 5 min at room temperature. A magnetic rack was used to capture the DNA binding beads. The beads were then washed twice using 200 μL of 80% ethanol, and DNA was eluted with 60 μL of EB buffer.

DNA extraction for C-circle assays was carried as previously described^{53 (link)}. Cells were lysed at 37 °C in 2% SDS, 50 mM Tris-pH 7.5, 20 mM EDTA and 200 μg/ml Pronase Protease (Sigma) followed by DNA concentration by Sodium-Acetate/Ethanol precipitation. DNA quantification was carried out using the Qbit dsDNA BR kit (thermo-fisher) and DeNovix DS-11 FX + spectrophotometer following the manufacturers recommendations. For C-circle reactions, 32 ng of extracted DNA was added to PCR master mix containing 0.2 mg/ml BSA, 0.1% Tween-20, 4 mM DTT, 1 mM each in the presence or absence of Phi-29 polymerase (NEB, 3.75U/32 ng DNA) and 1x Phi-29 reaction buffer (NEB) with rolling circle amplification carried out as previously described^{53 (link)}. To quantify C-circle abundance, each reaction was diluted with 100 μl 2X SSC and slot blotted using Bio-Rad Slot blot apparatus onto Hybond-N + membranes (Amersham) which were UV crosslinked with autocrosslink settings (120 mJ/cm²) of Stratalinker 1800 (Stratagene) apparatus, hybridised overnight at 37 °C with ³²P(CCCTAA)₃ labelled telomere probe in PerfectHyb buffer (Sigma) before washing 4× at 37 °C in 0.5X SSC, 0.1% SDS buffer and imaging using Typhoon FLA 9000 (GE Healthcare). Densitometric analysis was performed using FIJI software.