Population sequencing
Population sequencing
Illumina sequencing libraries were made directly from glycerol stocks of the original population samples, as well as the wild-type ancestral strain (GSY1136). Stocks were melted, and genomic DNA was extracted from 500 µl of each stock using Zymo Yeast Genomic DNA columns. The Nextera library prep kit (Epicentre) was used to construct the libraries, starting with 25–50 ng of genomic DNA. The tagmentation reaction was performed in LMW Reaction Buffer at 55°C for 10 minutes. The resulting tagged DNA was subjected to PCR using the Nextera PCR enzyme (Epicentre) under the following conditions: 72°C for 3 min, 95°C for 30 sec; 9 cycles of 95°C for 10 sec, 62°C for 30 sec, 72°C for 10 sec; final extension at 72°C for 1 min. A shortened extension time was used to bias the amplification of short fragments in order to maximize the proportion of bases being sequenced twice with overlapping paired-end Illumina reads. A modified Adapter 2 with a random hexamer barcode of sequence 5′-CAAGCAGAAGACGGCATACGAGATNNNNNNCGGTCTG​CCTTGCCAGCCCGCTCAG-3′ (PAGE-purified, IDT Technologies) was used during the PCR for the population samples, while the standard Nextera Adapter 2 was used for GSY1136. No size selection was performed on the libraries, although they were concentrated through a Qiagen MinElute column. The same GSY1136 library was spiked into all 24 population libraries at a molar rate of 5%. The resulting libraries were sequenced on one lane apiece of 2×101 bp plus a 6 bp index read on the Illumina Hi-Seq 2000. In addition, two independent libraries from the same genomic DNA of GSY1136 were sequenced on one Hi-Seq lane apiece.
Nextera
Population sequencing