Exome capture of T.turgidum cv Kronos: Green flag leaf bulk of Kronos TILLING line K2282, F2 population K2282-28
Pooled DNA from F2 plants with the green flag leaf phenotype (wild-type) was used for library preparation, following quality checks. library preparation was carried out with a KAPA HTP Library Prep Kit. Size selection was carried out using Beckman Coulter XP beads, and DNA was sheared to approximately 350 bp using the Covaris S2 sonicator. Four libraries were produced, one for each bulk detailed above, which were barcoded and pooled. Five cycles of PCR were carried out on the libraries before carrying out exome capture. Hybridization to the wheat NimbleGen target capture, previously described in Krasileva et al. (2017), was carried out using the SeqCapEZ protocol v5.0, with the following changes: 2.8 L of Universal Blocking Oligos was used, and the Cot-1 DNA was replaced with 14 L of Developer Reagent. Hybridisation was carried out at 47C for 72 hours in a PCR machine with a lid heated to 57C. The library pool was diluted to 2 nM with NaOH and 10L transferred into 990L HT1 (Illumina) to give a final concentration of 20pM.  This was diluted further to an appropriate loading concentration in a volume of 120 L and spiked with 1% PhiX Control v3 before loading onto the Illumina cBot. The flow cell was clustered using HiSeq PE Cluster Kit v4, utilising the Illumina PE_HiSeq_Cluster_Kit_V4_cBot_recipe_V9.0 method on the Illumina cBot. After clustering, the flow cell was loaded onto the Illumina HiSeq2500 instrument following the manufacturers instructions. The sequencing chemistry used was HiSeq SBS Kit v4.  The library pool was run on two lanes with 125 bp paired end reads. Reads in bcl format were demultiplexed using the 6 bp Illumina index by CASAVA 1.8, allowing for a one base-pair mismatch per library, and converted to FASTQ format by bcl2fastq.