Smart pooling
Smart pool construction  Frozen strain stocks in the Bioneer library were thawed at room temperature and pinned onto YES plate containing 150 mg/l G418 and 100 mg/l carbenicillin using a 96-pin replicator. Two days later, the strains were inoculated into 96-well deep well plates containing liquid YES medium plus G418 and grown for 3 days in a shaker. Cells in each well were dispensed into 5 different pools according to shifted transversal design of STD(3125;5;5), which can accommodate up to 3125 wells {Thierry-Mieg, 2006 #5}. A total of 25 pools were constructed with each pool containing cells from 563 wells.  Multiplex deep sequencing library preparation  Genomic DNA was extracted from each pool using the MasterPure Yeast DNA Purification Kit (EPICENTRE Biotechnologies). The barcodes in each pool were amplified with Ex Taq HS DNA polymerase through 30 cycles of 20 s at 94°C, 20 s at 53°C, and 20 s at 72°C. For uptags, the forward primer (upf-X) was 5’-CACGACGCTCTTCCGATCTXXXXGAGGCAAGCTAAGATATC-3’, the reverse primer (upr) was 5’-AGCAGAAGACGGCATACGAGCCTTACTTCGCATTTA-3’. For dntags, the forward primer (dnf-X) was 5’-CACGACGCTCTTCCGATCTXXXXCCAGTGTCGAAAAGTATC-3’, the reverse primer (dnr) was 5’-AGCAGAAGACGGCATACGATTGCGTTGCGTAGG-3’. “XXXX” in the forward primer sequences denotes the 4-digit multiplex indexing tags. Fifty tags were selected from a set of 64 tags that differ from each other by at least two substitutions. The PCR products were diluted 200 folds and used as template for another round of PCR to add sequences needed for Illumina sequencing. The cycling parameters were: 20 cycles of 20 s at 94°C, 20 s at 56°C, and 20 s at 72°C. The same pair of primers was used for all fifty PCR reactions. The forward primer (seqf) was 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’, the reverse primer (seqr) was 5’-CAAGCAGAAGACGGCATACGA-3’. The second round PCR products were gel purified and mixed together in equal molar ratios to use as the Illumina sequencing template. Standard single-end sequencing primer was used and 42 cycles of sequencing were carried out with Genome Analyzer II.