For Pfcytb sequence of parasites surviving drug pressure, lysates were prepared from cultures at 1% parasitemia (90 °C, 5 min), and a 1848 bp fragment (containing the 1131 bp Pfcytb open reading frame) was amplified with primers (SI Appendix , Fig. S2A ,B ) and Phusion DNA polymerase (NEB). PCR product was verified by gel electrophoresis as single band of predicted size, and sequenced. Sequences were aligned to P. falciparum 3D7 cytochrome b (PF3D7_MIT02300) using NCBI blast, and positions on all trace files were assessed in SnapGene.
For whole genome sequences to identify the causative mutation (42 (link)), DNA was isolated from 108 asexual parasites; QiAamp DNA Mini Blood kit, Qiagen), quantified (Qubit HS ds DNA assay (Qubit Flex Fluorometer, ThermoFisher)), and quality assessed by Genomic Screentape analysis (TapeStation 2200, Agilent). Barcoded libraries for DNA-Seq were synthesized from 100 ng DNA (Celero EZ-Seq kit with NuQuant, Tecan Genomics), quality assessed by High Sensitivity DNA Lab Chips (BioAnalyzer 2100, Agilent), quantified by Qubit HS dsDNA assay, and sequenced on Illumina’s MiSeq platform, (2 × 300bp v3 with 5% PhiX; JHMI Synthesis and Sequencing Facility). Using Partek Flow with sequence defaults, analyses included pre-alignment QA/QC, adaptor/read trimming, reference genome alignment to P. falciparum NF54 (PlasmoDB) using Bowtie 2, post-alignment QA/QC, FreeBayes variant calling, variant filtering (VarQual ≥ 30), and variant annotation based on precedence rules. Sequence files for WT parent and Y268S mutant were deposited (NCBI Sequence Read Archive, BioProject ID: PRJNA913198). To identify changes unique to atovaquone-resistant parasites, sequences were compared to that of the isogenic parent line, and then to those of seven other NF54-derived atovaquone-sensitive strains.
For whole genome sequences to identify the causative mutation (42 (link)), DNA was isolated from 108 asexual parasites; QiAamp DNA Mini Blood kit, Qiagen), quantified (Qubit HS ds DNA assay (Qubit Flex Fluorometer, ThermoFisher)), and quality assessed by Genomic Screentape analysis (TapeStation 2200, Agilent). Barcoded libraries for DNA-Seq were synthesized from 100 ng DNA (Celero EZ-Seq kit with NuQuant, Tecan Genomics), quality assessed by High Sensitivity DNA Lab Chips (BioAnalyzer 2100, Agilent), quantified by Qubit HS dsDNA assay, and sequenced on Illumina’s MiSeq platform, (2 × 300bp v3 with 5% PhiX; JHMI Synthesis and Sequencing Facility). Using Partek Flow with sequence defaults, analyses included pre-alignment QA/QC, adaptor/read trimming, reference genome alignment to P. falciparum NF54 (PlasmoDB) using Bowtie 2, post-alignment QA/QC, FreeBayes variant calling, variant filtering (VarQual ≥ 30), and variant annotation based on precedence rules. Sequence files for WT parent and Y268S mutant were deposited (NCBI Sequence Read Archive, BioProject ID: PRJNA913198). To identify changes unique to atovaquone-resistant parasites, sequences were compared to that of the isogenic parent line, and then to those of seven other NF54-derived atovaquone-sensitive strains.
