Identifying crossover locations in an Arabidopsis thaliana Col x Di-G F2 population using genotyping by sequencing

Genomic DNA from 239 Col x Di-G F2 individuals was extracted using CTAB and used to generate sequencing libraries as described (Rowan et al; Yelina et al), with the following modifications. DNA was extracted from 3 rosette leaves of 5 week old plants and 150 ng of DNA used as input for each library. DNA shearing was carried out for 20 minutes at 37°C with 0.4U of DNA Shearase (Zymo research). Each set of 96 libraries was sequenced on one lane of an Illumina NextSeq500 instrument (300-cycle Mid Output run). The barcoded adapter sequences were trimmed and bases with quality lower than 19 were filtered using Cutadapt. Sequencing data was analysed to identify crossovers as previously reported, using the TIGER pipeline.