Big dye terminator v3.1 cycle sequencing kit

Nested PCR products were purified with ExoSAP (New England Biolabs, Ipswich, MA, USA). The cycling sequencing was carried out using Big Dye Terminator v3.1 cycle sequencing kit on Bio-Rad C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). Dye terminators were cleaned by precipitating reactions with EDTA/NaOAc/Ethanol precipitation and rehydrating in 10 μl HiDi formamide. Capillary electrophoresis was performed in ABI 3730 or 3730xl genetic analyzer (Applied Biosystems, Foster City, USA). Sequence data were analysed using the Geneious Prime R10 and R11 (Biomatters, San Francisco, CA, USA). The mixed SNPs were identified where the minor peak was ≥ 30% of the major peak. SNPs at Pfdhfr, Pfdhps, Pfcrt, Pfmdr1 genes and Pfk13 propeller region were identified by comparing sequences with reference 3D7 strains from GenBank XM_001351443.1, Z30654.1, XM_001348968.1, XM_001351751 and MW712622.1), respectively. PCR reaction systems and cycling conditions were detailed in Additional files 1, 2.

Paired pre-treatment and day of recurrent parasitaemia samples were assessed for known markers of resistance in the Pfcrt, Pfk13, and Pfmdr1 genes. In addition, all pre-treatment samples were assessed for mutations in the Pfk13 and Pfmdr1 genes. Fragments of Pfk13, Pfcrt, and Pfmdr1 were amplified by nested PCR using previously published primers [28 (link), 29 (link)]. Direct Sanger sequencing of the nested purified PCR products was performed by using a BigDye Terminator v3.1 cycle sequencing kit on an iCycler thermal cycler (Bio-Rad, California, USA). Sequence analysis was performed using Geneouis R7 (Biomatters, Auckland, New Zealand). The Pfcrt codons 72, 74, 75, and 76, Pfk13 propeller domain (codon positions: 389–649), and Pfmdr1 codons 86, 184, and 1246 were analysed for SNPs. The P. falciparum laboratory strain 3D7 Pfcrt, Pfk13, and Pfmdr1 were used as reference sequences for the analysis. Molecular analyses were performed in collaboration with the U.S. Centers for Disease Control and Prevention (CDC) Malaria Laboratory in Atlanta, USA, as part of the President’s Malaria Initiative (PMI)-supported Anti-malarial Resistance Monitoring in Africa Network [30 (link)].