Similar to the standard Illumina library construction protocol, the sheared DNA is subjected to end repair and 3′-end dA-tailing. Notably, in our initial description of DS19, we used A-tailed adapters and enzymatically added a 3′ dT overhang on the fragmented DNA sample. However, since then, we have determined enzymatic T-tailing to be considerably less efficient than A-tailing (Supplementary Fig. 5), which results in a substantially reduced number of final reads. We have re-engineered the adapters to have a 3′ dT overhang (Fig. 1a, see ‘Adapter synthesis’ section and Boxes 1 and 2), which simultaneously increases the final data yield (Supplementary Table 1) and makes the protocol more consistent with conventional NGS library preparations.
