Whole Blood DNA Isolation and SWGA

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.