The DNA:adapter ratio is a crucial variable, as too few adapters lead to inefficient ligation, and the use of excess adapters can result in adapter dimers, which, owing to their small size, preferentially amplify during PCR. The presence of these adapter dimers interferes with DNA sequencing. To minimize the presence of adapter dimers, we determine the molar concentration of DNA molecules in the preligated DNA library on an Agilent TapeStation 2200 or Bioanalyzer 2100 and then use 20-fold molar excess of adapter relative to DNA. Unligated adapters and adapter dimers are then removed by purification with Ampure XP beads (Supplementary Fig. 6).
