Amplicon Sequencing of P. falciparum Drug Resistance Genes and Vaccine Candidate antigens: Isolate 5
DNA was extracted from blood samples collected in EDTA vacutainers, using QIAamp DNA Blood Mini or Midi Kit (Qiagen Inc., catalog nos. 51106, 51185). Gene Specific Primers were used to amplify targets. For each sample, 10 ul of each amplicon was pooled, the pooled mixture purified using a QIAquick PCR Purification Kit (Qiagen Inc., catalog no. 28106) and quantitated using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, catalog no. Q32866). Barcoded sequencing libraries were generated from 100-500 ng of purified pooled PCR product using ion expressTM barcode adapters (Life Technologies, catalog no. 4471250 for barcodes 1-16; catalog no. 4474009 for barcodes 17-32) and library prep kit (New England Biolabs, catalog no. E6285L). The size and quality of libraries were determined using an Agilent bioanalyzer, and quantified by quantitative PCR using Ion Torrent Library Quantification kit (KAPA biosystems, KK4857,) on a LightCyclerR 480 (Roche). For most experiments, 16 barcoded libraries were pooled for a single run on an Ion Torrent Personal Genome Machine using Ion PGMTM Hi-QTM sequencing technology and IonTM 314v2 type chips (one library pool was run on an IonTM 318v2 chip).