Selective Whole Genome Amplification for P. falciparum

The detailed methodology for detecting and sequencing P. falciparum genome using selective whole genome amplification (sWGA) has been published by Aninagyei et al [24 (link)]. The sWGA reaction was performed using DNA template (≥ 5 ng). The reaction mix was made up of the following: template DNA, 1× bovine serum albumin, 1 mM dNTPs, 2.5 μM of each amplification primer, 1× Phi29 reaction buffer and 30 units of Phi29 polymerase enzyme (New England Biolabs). Isothermal amplification conditions were used (35 °C for 5 min, 34 °C for 10 min, 33 °C for 15 min, 32 °C for 20 min, 31 °C for 30 min, 30 °C for 16 h prior to denaturing Phi29 polymerase enzyme 65 °C. Ampure XP cleaned amplicons washed with 200 μL of 80% ethanol and eluted with elution buffer. Using the NEBNext® Ultra™ DNA library preparation kit (New England Biolabs), DNA libraries were prepared prior to sequencing on Illumina HiSeq 2500 DNA Sequencer. After sequencing, demultiplexing and fastq data files were generated automatically. Low quality reads were trimmed (Bioedit v7.2) and each dataset analysed independently by mapping sequence reads to the P. falciparum 3D7 reference genome using Burrows-Wheeler Aligner. Allelic analysis was done for Pfcrt, Pfdhfr, Pfdhps, Pfmdr1 and Kelch13 genes.