title: S695P
design: An msh2 knockout strain was transformed with centromere-based plasmids harboring variants of msh2, wild-type msh2, or the vector. The strains were propagated in synthetic medium lacking histidine to select for the plasmids. A single colony from each transformation was selected to begin the mutation accumulation experiment. Strains were passaged on synthetic medium lacking histidine for ~170 generations with bottlenecks every ~21 generations. The bottlenecks were accomplished by picking a single colony, streaking for single colonies approximately every two days, repeating the process eight times.  In preparation for sequencing, a single colony was selected and grown in 25 ml YEPD (yeast extract, peptone, dextrose) medium supplemented with adenine (Burke et al. 2000) until saturation was achieved (24-40 h). Genomic DNA preparations from yeast were as described previously (Burke et al. 2000) except the glass bead lysis step was accomplished with a Fastprep®-24 instrument (MP Biomedicals LLC).Yeast genomic DNA was prepared for sequencing using the Illumina TruSeq™ DNA Sample Preparation kit with six indices for multiplexing.   Burke, D., D. Dawson, T. Stearns and Cold Spring Harbor Laboratory., 2000 Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y. 