Duplex Sequencing was initially described with use of A-tailed adapters10,19; we have since found that T-tailed adapters result in improved ligation efficiency, and have published a detailed protocol for their synthesis and use20. In brief, DNA was sheared, end-repaired, and A-tailed, then ligated to T-tailed Duplex Sequencing adapters using a 20x molar excess of adapters relative to A-tailed DNA molecules. Following reaction cleanup with 1.0 volumes of Ampure XP beads (Agencourt), the adapter-ligated DNA was PCR amplified for 5 cycles with the KAPA Biosystems hot start high-fidelity kit, using primers mws13 and mws20. 240 nanograms of input DNA was used in each 100 microliter PCR reaction, with two to eight PCR reactions performed per sample. Due to the small amount of on-target DNA present in the starting sample, multiple PCR reactions are needed to amplify sufficient on-target DNA for capture. Each PCR reaction results in sequence data representing approximately 500 independent genomes; the number of PCR reactions performed can be adjusted depending on the sequencing coverage desired. The products from all reactions were pooled and purified with 1.2 volumes of Ampure XP beads, with a final elution volume of 50 microliters.
