Phi29 polymerase enzyme

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.

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.

The step-wise method for the SWGA has been published in a separate study [20 (link)]. In summary, genome amplification reaction was performed in 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). Amplification conditions were using step down temperatures from 35°C to 30°C with increasing incubation time from 5 mins to 16 hours. The polymerase enzyme was inactivate at 65°C prior to cooling to 4°C.