title: Vibrio cholerae Delta_crtS_D185-EV; exponential culture sample
design: This sample resulted from a Short-Term Evolution Experiment and was intended for Marker Frequency Analysis (MFA) and mutation detection. In this strain the locus crtS was deleted from the large chromosome, chr1. Replication of the crtS locus triggers replication of the small chromosome, chr2. Cells were grown exponentially in Mueller Hinton medium (MH) at 37°C under agitation. Each culture of mutant strains was diluted in fresh medium every morning and every evening, allowing them to grow seven generations during the day and nine generations overnight. Based on the volume of media in the flasks (as Volume = 2**n, with n = # of generations of bacteria), and to minimize bottleneck effects, 1 ml of the previous culture is added to the fresh medium, with a 1:128 dilution factor for day growth and a 1:512 dilution factor for overnight growth. The extended time allowed for growth allows the slowest-growing mutant to reach stationary phase before proceeding to the next dilution. Genomic DNA was prepared from exponentially growing cells harvested at OD450 ~0.15. Sequencing libraries were prepared for sequencing by automat, targeting an insert size of 200 base pairs bp. Libraries were sequenced using an Ion Proton sequencer PI chip. MFA was performed essentially as described in Skovgaard et al. (2011) PMID: 21555365; doi: 10.1101/gr.117416.110.