Illumina HiSeq 2000 paired end sequencing
Genomic DNA was extracted from homozygous (monosporic derivative) yeast strains as described in Bensasson (2011). The Genomic Technologies Core Facility at the University of Manchester generated high-throughput short-read paired-end sequence for genomic DNA from each strain. More specifically, DNA was quantified on a Qubit fluorimeter, and 50 ng DNA for each sample was simultaneously tagged and fragmented (tagmented) using the Nextera DNA sample preparation kit (Illumina). Following a cleanup using the ZymoTM Purification Kit (ZR-96 DNA Clean & ConcentratorTM-5 from Zymo Research), the purified, tagmented DNA was then amplified via limited-cycle PCR which also added the indices (i7 and i5) and sequencing primers. AMPure XP beads were then used to purify and size select the library DNAs. The libraries were then normalized to 2nM and pooled prior to cluster generation using a cBot instrument. The loaded flow-cell was then paired-end sequenced on an Illumina HiSeq2000 instrument. Demultiplexing of the output data (allowing one mismatch) and BCL-to-Fastq conversion was performed with CASAVA 1.8.3
UNIVERSITY OF MANCHESTER
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