Phi29 dna polymerase enzyme

Thirty to 70 ng of input DNA was added to a 50-µl reaction mixture containing 3.5 µM SWGA primers, 30 U phi29 DNA polymerase enzyme (New England Biolabs), 1× phi29 buffer (New England Biolabs), 4 mM dNTPs (Roche), 1% bovine serum albumin, and water. The reaction was carried out on a thermocycler with cycling conditions consisting of a ramp down from 35°C to 30°C (10 min per degree), 16 h at 30°C, 10 min at 65°C, and hold at 4°C. The samples were diluted 1:1 with DNase-free, RNase-free water and purified with AMPure XP beads (Beckman-Coulter) at a 1:1 ratio according to the manufacturer’s protocol. When a second round of selective amplification was performed, the reaction mixture contained 100 to 200 ng of the AMPure XP-purified product from the first reaction.

DNA was isolated from thawed whole blood using QIAamp DNA Blood Mini Kit (Qiagen) following the manufacturer’s recommendation and as described elsewhere [17 (link)]. Samples were subsequently resuspended in TE buffer, and genomic DNA was quantified using a Qubit 2.0 fluorometer (ThermoFisher). Thirty to 70 ng of input DNA was added to a 50-μl reaction containing 3.5 μM SWGA primers, 30 U phi29 DNA polymerase enzyme (New England Biolabs), phi29 DNA buffer (New England Biolabs), 1% bovine serum albumin, and water as previously described [18 (link), 19 ]. The primer set used consists of 12 primers: 5′-AACGAAGC*G*A-3′, 5′-ACGAAGCG*A*A-3′, 5′-ACGACGA*A*G-3′, 5′-ACGCGCA*A*C-3′, 5′-CAACGCG*G*T-3′, 5′-GACGAAA*C*G-3′, 5′-GCGAAAAA*G*G-3′, 5′-GCGAAGC*G*A-3′, 5′-GCGGAAC*G*A-3′, 5′-GCGTCGA*A*G-3′, 5′-GGTTAGCG*G*C-3′, and AACGAAT*C*G. The reaction was carried out on a thermocycler and consisted of a ramp down from 35 to 30 °C (10 min per degree), 16 h at 30 °C, 10 min at 65 °C, and hold at 4 °C. The samples were diluted 1:1 with DNAse-free and RNAse-free water and purified with Ampure XP beads (Beckman-Coulter) at a 1:1 ratio per the manufacturer’s protocol.

Monoinfection samples were sequenced using whole genome amplification as previously described^{20 (link)}. Briefly, we performed two amplification rounds with 50 ng of input DNA to a 50ul reaction mixture containing 5 μM SWGA primers (set 6A and 8A). Each amplification mix contained 30U phi29 DNA polymerase enzyme (New England Biolabs), phi29 DNA buffer (New England Biolabs), 1% bovine serum albumin, 1 mM dNTPs and water. The amplification reaction was carried out on a thermocycler and consisted of a ramp down from 35 to 30 °C during 10 min per degree, 16 h at 30 °C, 10 min at 65 °C and hold at 4 °C.
Amplified samples were diluted in 1:1 molecular grade water and purified using the Ampure XP (Beckman-Coulter) beads system at a ratio of 1:1 as per manufacturer’s protocol. Paired-end sequencing libraries were generated using 1 ng amplified DNA using the Nextera XT kit (Illumina, California, USA) following the manufacturer’s protocol. The pooled library was sequenced using the MiSeq reagent kit v3 (600 cycles) and MiSeq reagent kit v2 (500 cycles) and sequenced on an Illumina MiSeq sequencer to generate 250–300 bp paired end reads.