Nimblegen 12 135 k array

To select probes for the construction of a secondary array, CS, M808, and 14 randomly selected RILs were genotyped. After digestion of total DNA with PstI and BstNI, adapter ligation, PCR amplification and purification of PCR products as described in the library preparation section, they were labelled with Cy3 using the NimbleGen One-Color DNA Labeling Kit (Roche Diagnostics) according to the manufacturer's instructions. Hybridization was performed at 42°C for 72 h on a NimbleGen Hybridization System (Roche Diagnostics). Arrays were scanned using a NimbleGen MS200 Microarray Scanner (Roche Diagnostics) and genotype calling was performed based on the signal intensities.
For selection of probes, a χ² test was used to compute segregation of each probe in the 14 RILs. Those deviating from Mendelian 1 : 1 segregation at a 1% significance level were discarded. The secondary array was constructed using one selected probe per contig or read, consisting of 21,346 CS-derived probes, 21,205 M808-derived probes, and 2,303 probes for normalization. These 44,854 probes were spotted in triplicate on a NimbleGen 12 × 135 K array (Roche Diagnostics). This array was used for genotyping of 210 RILs and the two parental lines.

Molecular karyotyping of DNA was performed with the Illumina HumanCytoSNP-12 (version 2.1) or Infinium CoreExome-24 arrays, as previously described (Bruno et al., 2011 (link)). Automated detection of long contiguous segments of homozygosity was performed with the CNVPartition v3.1.6 algorithm in KaryoStudio software. SNP genotypes were generated in GenomeStudio software (Illumina) with data from a set of 102 intra-run samples.
A custom comparative genomic hybridization (CGH) NimbleGen 12 × 135 K array (Roche Diagnostics) was designed to densely tile 1034 MitoExome genes encoding known mitochondrial proteins (Calvo et al., 2012 ). Targeted exonic regions were tiled to an average probe spacing of 50 bp, intronic regions to an average of 900 bp, and regions directly upstream and downstream of targeted genes were covered forming a low-resolution backbone tiled at 3600 bp. CGH arrays were performed, scanned and analysed in accordance with manufacturer’s recommendations using gender matched, pooled DNA from seven unaffected individuals as a control.
Intragenic deletions were further investigated by long-range PCR amplification and sequencing of the junction region (BigDye® v3.1 terminators; Applied Biosystems) to better define the breakpoints. The primer sequences used in this study and their binding sites within the ATAD3 region are shown in Supplementary Fig. 1.