Selective Amplification of P. falciparum Genome

Detailed P. falciparum primers designed to identify P. falciparum genes have been published (Oyola et al., 2016 (link)). Selective whole genome amplification (sWGA) reaction was performed in a 50 μL reaction volume containing at least 5 ng of template DNA, 1× BSA, 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 conditions were used for sWGA. The conditions were 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 then heating at 65 °C for 15 min to inactivate the enzymes prior to cooling to 4 °C. Subsequent to sWGA, the products were cleaned using Ampure XP beads after which the bead/DNA mixture was placed on a magnetic rack to capture the DNA-bound beads. Beads were washed twice with 80% ethanol and the bound DNA eluted with elution buffer. DNA libraries were prepared with the cleaned DNA products using NEBNext DNA sample preparation kit (New England Biolabs). DNA libraries were sequenced using Illumina HiSeq 2500 DNA Sequencer.