Multiplex MDS Library Preparation

MDS was performed as previously described (Jee et al., 2016 (link)). In brief, samples of 10 ml from the last cultures of the four B. subtilis strains were spun down and resuspended in 1 ml Tris-EDTA buffer (pH7.5) and incubated −80°C overnight. Genomic DNA extraction was performed using Qiagen genomic tip (100G) and quantified using Nanodrop. To generate each library, 2 μg of genomic DNA were independently treated with NlaIII (leading strand) or Hpy166III (lagging strand) restriction enzyme, which cleaves and delimits the Region Of Interest (ROI). The primers design was performed considering the cut sequence of NlaIII or Hpy166III for leading or lagging strand, respectively. A single PCR cycle was performed with 500 ng of restriction enzyme treated genomic DNA, 500 μM barcoded forward adapter primers annealing to the 3′ end of the ROI and Q5 Hot Start polymerase (New England Biolabs; Ipswich, MA, United States). Unused barcodes were removed with ExoI. The forward library was amplified using a forward adapter (described below in “Primer schema”) and performing 14 cycles of PCR with Q5 Hot Start high-fidelity DNA polymerase. Reverse adapter primers were used to define the ROI. Finally reverse adapters were used to amplify the libraries in 15 cycles of PCR. All libraries were resolved in an 8% acrylamide gel and purified with Ampure XP beads (see Supplementary Figures S2, S9).