E gel ex agarose 2

The BFG‐GI technology relies on the Cre/Lox system to recombine the complementary donor and recipient loxP/lox2272 sites that serve to introduce the donor barcode adjacent to the recipient barcode (Fig 1). We multiplex‐sequenced the fused barcodes from pools of cells using the following steps: (i) genomic DNA extraction using glass beads and phenol/chloroform; (ii) PCR amplification of the 325‐bp barcode fusion product including the two 20‐bp barcodes and the multiplexing sequencing adapters (one index for each condition, for each technical replicate); (iii) concentration and gel purification of amplicons using 2% E‐Gel EX agarose 2% (Invitrogen), DNA Clean & Concentrator Kit (Zymo Research), and MinElute Gel Extraction Kit 50 (Qiagen); (iv) normalization of DNA libraries using Qubit Fluorometric Quantitation (Invitrogen); (v) mix of libraries at equal concentrations; (vi) quantification of the pooled DNA library mix by qPCR; and (vii) sequencing by Illumina 75‐cycle NextSeq paired‐end technology, including 25 cycles for each barcode and 6 cycles for the multiplex index. We mapped sequencing *.fastq files against the library of expected barcode sequences using the program Segemehl (v0.1.7, ‐A 85) and custom scripts; 97% of all sequencing reads mapped to expected barcodes.

Ten strain pairs with one strain with GIS r < 0.5 and another with GIS r > 0.5 with other replicates for the same gene were selected for genome sequencing. Genomic DNA from 20 strains was extracted via cell wall disruption with Zymolyase 100T 10 mg/ml (Amsbio) and purification using AMPure beads (Agilent). gDNA was quantified with Quant‐iT PicoGreen dsDNA assay kit (Invitrogen) and normalized to 2 ng/μl for DNA fragmentation and library normalization with a Nextera XT DNA Library Prep Kit, using a transposase (Tn5) for tagmentation. A limited‐cycle PCR was used to add Illumina sequencing adapters and indices i5 and i7. PCR amplicons with size between 400 and 800 bp were gel‐purified using a 2% E‐Gel EX agarose 2% (Invitrogen) and MinElute Gel Extraction kit (Qiagen). Whole‐genome sequencing was conducted on an Illumina NextSeq 500 using a HighOutput 150 cycles v2 kit with 40× coverage. Sequencing results were mapped against the reference genome UCSC sacCer3 (SGD vR64.1.1), corrected for GC content, and chromosomal duplications detected with the HMMcopy R package (Ha et al, 2012).