For the 100 bp libraries, 100 ng of genomic DNA was sheared by adaptive focused acoustics using a Covaris S2 (Covaris Inc.) and Covaris Micro-tubes and using the method and shearing conditions described in the Ion Fragment Library Kit (publication 4467320 Rev. B) protocol. For 200 bp libraries, 200 ng of genomic DNA was also sheared using a Covaris S2, with modification of the shearing conditions (intensity, 4; time, 27 sec). The remaining steps in the library preparation were performed using the Ion Plus Fragment Library Kit, using the corresponding User Guide (Publication 4471989 Rev. B), with modification. Due to the shearing volume required for the Covaris S2 micro-tubes, the sheared DNA samples were treated as if they were 1 µg samples, in the end repair steps, as this catered for a larger starting volume. From that point forward the library was treated as if the input was 100 ng. Following adapter ligation and nick repair, the library was size selected using a Pippin Prep (Sage Science) instrument, followed by 6 cycles of amplification.
