Copy-number analysis in duplication regions

Semi-synX(A-C), semi-synX(A-E), synX(A-R), and control (BY4741) strain were grown overnight at 30°C in 5 ml of YPD. Genomic DNA was prepared following the protocol “Yeast genomic DNA preparation for DNA sequencing.” DNA was eluted in 50 μl of water. Primers were designed by using the PrimeQuest Design tool on the IDT website and are listed in table S6. Primers were selected to anneal outside PCRTag sequences and, thus, amplify both synthetic and native DNA. qPCR reactions were carried out in a final volume of 4 μl by using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad; 172-5272). Each reaction included 75 nl of cDNA and 50 nl of forward/reverse primers mix (50 μM each) introduced into each well of a Hard-Shell Thin-Wall 384-Well Skirted PCR Plate (Bio-Rad; HSP- 3905) by using the Echo (LabCyte). Gene expression analysis was performed by using the CFX Software Manager (Bio-Rad). qPCR was performed on technical triplicates and data were analyzed by using two reference genes (TAF10 and UBC6).