Lane 1-3 with pooled strains
Bacterial genomic DNA preparations from 32 strains were isolated using QIAamp DNA mini kit (Qiagen)  with RNase treatment. Isolated genomic DNA was fragmented by sonication using Bioruptor (Diagenode)  to an average size of 100-500bp and con?rmed by gel electrophoresis. 2 µg aliquots of fragmented genomic  DNA from each strain were mixed to create genomic DNA pools. There were 10 pools created. Of these,  8 pools contained 3 strains and 2 pools containing 4 strains such that each strain was in only one pool.  10 tagged genomic sequencing libraries were constructed using the Multiplex Sample Prep Oligo Kit  following protocols provided by the manufacturer (Illumina). A mean library fragment size of 200-250bp  (mean insert size of 100-150bp) was achieved by gel puri?cation relative to standard sized markers. The  puri?ed library size distribution was con?rmed by capillary electrophoresis in a Bioanalyzer (Agilent).  10 libraries were mixed in a proportion to maintain an equal amount of DNA from each strain in the  multiplexed sequencing library. Final library concentration was determined by ?uorescent based assay  on the Qubit (Life Technologies). 7pmol of each mixed library was loaded onto each ?ow cell.