Genotyping module

1,580 Prunus accessions were genotyped using the International Peach SNP Consortium (IPSC) peach 9K SNP array v1 [17 (link)]. SNP genotypes were scored with the Genotyping Module of the GenomeStudio Data Analysis software (Illumina, Inc.) using the default parameters.
The SNPs were divided into five categories: A, B, C, D, and E. The first three included the polymorphic SNPs and the last two the non-polymorphic. The SNPs with GenTrain higher than 0.4 and GeneCall 10% higher than 0.2 and at least two genotypic classes were classified as polymorphic. The three classes of polymorphic SNPs were defined as follows:
The non-polymorphic SNPs were divided as:
All further analysis were performed using all Class A SNPs with a minor allele frequency (MAF) higher than 0.05. Genotypic data have been uploaded to GDR database (http://www.rosaceae.org/) under the accession number tfGDR1013 and in the FruitBreedomics database (http://bioinformatics.tecnoparco.org/fruitbreedomics/).

Illumina SNP-array data were previously obtained from 30 samples using a HumanCytoSNP-12 v2.1 beadchip on an iScan system, following standard protocols as provided by the manufacturer (Illumina). SNP genotypes were extracted from Illumina GenomeStudio (v2011.1) using the Genotyping module (v.1.9.4). Concordance between NGS and SNP-array genotypes was defined as identical calls with both techniques.

Raw fluorescence intensities were loaded into GenomeStudio 2.0 with Genotyping Module (Illumina, Inc.) in order to do Quality Control. Six thousand one hundred ninety (6,071 controls, 119 cases) samples with Call Rate above 0.95 were included into further analysis. Variants with incorrect clustering, Multi-allelic SNPs, and polymorphisms located at X and Y chromosomes were excluded from the analysis. 323,583 remaining SNPs where further processed in PLINK 1.9 software (20 (link)). One hundred fifty-six thousand three hundred forty-one variants were removed due to minor allele threshold and an additional 2,847 variants were removed due to Hardy–Weinberg exact-test (21 (link)). Therefore, statistical analysis was carried out on 164,395 variants. StrandScript was then used to ensure forward strand orientation (22 (link)).

A total of 1899 individuals from the two cohorts were genotyped by the Illumina Infinium HumanCoreExome Beadchip platform (Illumina, San Diego, CA). Genotypes were called using the Genotyping module (version 1.9.4) of GenomeStudio software (version 2011.1, Illumina). Closely related individuals, individuals with an extreme inbreeding coefficients, individuals with mislabeled sex, individuals with a call rate < 95%, duplicates and individuals identified as ethnic outliers, as well as genetic markers with a call rate < 98%, a minor allele frequency < 0.01 and a Hardy–Weinberg equilibrium P < 1 × 10^–5, were removed during quality control. Imputation was performed on the Michigan imputation server [12 (link)] using haplotypes from the Haplotype Reference Consortium (HRC version r1.1) panel [13 (link)].

MommeD44 heterozygous mice were backcrossed twice to Line3C (see above) and phenotyped for GFP expression by flow cytometry. DNA from tail tissue collected during flow cytometry procedures was used to perform linkage analysis. The Illumina GoldenGate genotyping assay (Mouse Medium Density Linkage Panel) was used with 10 wildtype and 13 heterozygous mice. MommeD44 wildtype samples should only have heterozygous C57BL/6J SNPs surrounding the causative mutation and MommeD44 mutants should have FVB and C57BL/6J SNPs at this interval. The Mouse Medium Density Linkage panel contains 766 measurable SNPs between C57BL/6J and FVB/NJ. Samples were genotyped following the Illumina protocol and genotype calls were made using the Genotyping module of the GenomeStudio v1.1 software. Only samples with a call rate >95 were accepted. The linked interval was identified based on a peak in the LOD score. Fine mapping was carried out using primers amplifying C57BL/6J or FVB SNP loci that could be cut with restriction enzymes to determine genotype. Of the 95 F2 backcrosses, 8 mice had SNP profiles that were inconsistent with the mapping and were excluded. This small (<10%) error rate in phenotyping is commonly encountered in this screen [4 (link)].

Whole genome genotyping (WGG) data were available for 23 T-ALL patients (Figure 1). Normal and tumor samples from these patients were genotyped using Illumina's HumanOmni 2.5-Quad or HumanOmni2.5-Octo SNP bead arrays (McGill University and Genome Quebec Innovation Centre, Montreal, Quebec). Extracted genomic DNA was processed according to the Illumina Infinium HD Assay Ultra protocol. BeadChips were imaged on Illumina's iScan System with iScan Control Software (v3.2.45). The Genotyping Module (Version 1.9.4) of the Illumina GenomeStudio software (V2011.1) was used for raw data normalization, genotype clustering and calling, with default. ASCAT version 2.2 [103 (link)] was used to evaluate the sample purity, to evaluate tumor ploidy and to identify tumor-specific copy number variants (CNVs) or copy-neutral loss of heterozygosity (LOH).
CDKN2A gene allelic status was further evaluated in all T-ALL patients at diagnosis using PCR. Each 25 μl reaction contained 50 ng of template DNA, 1X KOD Buffer, 1.5 mM MgSO4, 200 μM dNTPs, 0.3 μM of each primer (listed in Supplementary Table S5) and 0.5U of KOD Hot Start DNA Polymerase (Millipore). Cycling parameters used were: 95°C 2 min; 40 cycles (95°C 20 sec, 58°C 10 sec, 70°C 10 sec). Amplified fragments of 368 bp were visualized using standard gel electrophoresis. Electrophoresis gels are available upon request.