WGS of S. cerevisiae: recombinant offspring of BY4741xRM11.1a FACS sorted for extreme high / low UPS activity
We used genomic DNA from our segregant pools to prepare a short-read library for whole-genome sequencing on the Illumina Next-Seq platform.  The Nextera DNA library kit (Illumina) was used according to the manufacturers instructions with the following modifications. We fragmented and added sequencing adapters to genomic DNA by adding 5 ng of DNA to a master mix containing 4 L of Tagment DNA buffer, 1 L of sterile molecular biology grade water, and 5 L of Tagment DNA enzyme diluted 1:20 in Tagment DNA buffer and incubating this mixture on a SimpliAmp thermal cycler using the following parameters (Thermo Fisher Scientific): 55 C temperature, 20 L reaction volume, 10 minute incubation.  We PCR amplified libraries prior to sequencing by adding 10 L of the tagmentation reaction to a master mix containing 1 L of an Illumina i5 and i7 index primer pair mixture, 0.375 L of ExTaq polymerase (Takara), 5 L of ExTaq buffer, 4 L of a dNTP mixture, and 29.625 L of sterile molecular biology grade water.  To multiplex samples for sequencing, we generated all 96 possible index oligo combinations using 8 i5 and 12 i7 index primers.  Libraries were PCR amplified on a SimpliAmp thermal cycler using the following parameters: initial denaturation at 95 C for 30 seconds, then 17 cycles of 95 C for 10 seconds (denaturation), 62 C for 30 seconds (annealing), and 72 C for 3 minutes (extension).  The DNA concentration of each reaction was quantified using the Qubit dsDNA BR assay kit (Thermo Fisher Scientific).  We pooled equimolar amounts of each sample, ran this mixture on a 2% agarose gel, and extracted and purified DNA in the 400 bp to 600 bp region using the Monarch Gel Extraction Kit (NEB) according to the manufacturers instructions.The pooled library was submitted to the University of Minnesota Genomics Center (UMGC) for quality control assessment and Illumina sequencing.  UMGC staff performed three quality control (QC) assays prior to sequencing.  The PicoGreen dsDNA quantification reagent (Thermo Fisher Scientific) was used to determine library concentration, with a concentration  1 ng/L required to pass.  The Tapestation electrophoresis system (Agilent Technologies) was used to determine library size, with libraries in the range of 200 to 700 bp passing.  Finally, the KAPA DNA Library Quantification kit (Roche, Basel, Switzerland) was used to determine library functionality, with libraries requiring a concentration  2 nM to pass.  The submitted library passed each QC assay.  The library was sequenced on a Next-Seq 550 instrument in mid-output, 75 bp paired-end mode, generating 153,887,828 reads across all samples, with a median of 9,757,090 and a range of 5,994,921 to 14,753,319 reads per sample. The mean read quality for all samples was > 30.