(iii) PCR amplification and sequencing of P. falciparum cytochrome b.

PCR primers were designed for amplification based on the annotated P. falciparum 3D7 cytochrome b gene sequence (MAL_MITO_3) on Plasmodb.org v27 as follows: Pf-cytb-PCRFOR, 5′-TGCCTAGACGTATTCCTG-3′, and Pf-cytb-PCRREV, 5′-GAAGCATCCATCTACAGC-3′. PCRs were amplified using the Phusion HS II High-Fidelity PCR master mix (ThermoFisher Scientific) with ∼20 ng parasite gDNA template according to the manufacturer's instructions with the following program: 98°C for the 30-s initial denaturation step, followed by 35 cycles of 98°C for 10 s, 54°C for 40 s, and 72°C for 30s and a final extension of 72°C for 7 min. PCR products were confirmed as a single, discrete band of 1,382 bp in length on a 1% agarose gel and then subsequently purified using the Qiagen PCR purification kit according to the manufacturer's instructions. Purified PCR products were prepared for Sanger sequencing service at Genewiz (Genewiz, South Plainfield, NJ) using the following sequencing primers: pf-cytb-SEQFOR1, 5′-GTGGAGGATATACTGTGAGTG-3′; pf-cytb-SEQFOR2, 5′-TACAGCTCCCAAGCAAAC-3′; pf-cytb-SEQREV1, 5′-GACATAACCAACGAAAGCAG-3′; and pf-cytb-SEQREV2, 5′-GTTCCGCTCAATACTCAG-3′. PCR primers Pf-cytb-PCRFOR and Pf-cytb-PCRREV were also used for sequencing purposes.