WGS of S. cerevisiae parental
Illumina library preparation was done using the Nextera DNA Flex Library Prep Kit (Illumina, 20018704) according to the manufacturers instructions. DNA was fragmented and tagged with adapter sequences using bead-linked transposomes. The tagged DNA, still bound to the bead-linked transposomes, was purified. Illumina Nextera DNA CD Index was added to individual libraries by PCR (five cycles). The indexed libraries were purified using solid phase reversible immobilization. After purification of the individual indexed libraries, the concentration of dsDNA in each library was quantified using the fluorometric Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, Q33231). The size of the libraries was verified using the Agilent 2100 Bioanalyzer DNA 1000 Kit (Agilent, 5067- 1504). The concentration of the amplifiable Illumina adapter-ligated molecules in the pooled library was established by qPCR using the KAPA Illumina Library Quantification Kit (Roche, KK4873  07960336001). The overall fragment size for the pooled library was determined using the Agilent TapeStation D5000 High Sensitivity Kit (Agilent, 5067-5593). The size adjusted library concentration was calculated, and the library diluted to 4 nM.The 4 nM pooled sequencing library was denatured using 0.2 N NaOH, diluted to 12 pM as recommended by the Nextera DNA Flex Library Prep Reference Guide, and combined with the denatured PhiX positive control at a spike-in concentration of 1% v/v. The denatured library was loaded on the Illumina MiSeq instrument and sequenced using the MiSeq Reagent Nano Kit v3 (600 cycles) (Illumina, MS-102-3003). The sequencer was programmed to perform a paired-end, dual indexed 2 X 301 cycle sequencing run. FASTQ files were automatically generated at the end of the run, saved on the MiSeq, the quality of the sequence run was analysed using Sequence Analysis Viewer (Illumina).