Genomestudio software

CpG methylation analysis was performed using Infinium 850k Methylation array (Illumina) according to the manufacturer’s recommendations. Briefly, 200 ng of DNA extracted from each sample was utilized to restore the degraded FFPE DNA to a state that is analyzable by the Infinium HD FFPE whole genome amplification workflow as described in the Infinium FFPE Restoration guide (Illumina). DNA was denatured using 8 µL of NaOH 0.1N for 10 min at room temperature. A 1 h reaction at 37°C was then performed with Primer Pre-Restore (PPR) and Amplification Mix Restore (AMR) reagents supplied by the kit manufacturer in which DNA repair is accomplished. Restored DNA was cleaned using a ZR-96 DNA Clean and Concentrator-5 kit (Zymo Research) following the manufacturer’s protocol. DNA was concentrated between 150 ng/uL of concentration. Between 100 and 200 ng of prepared DNA was used as input for the hybridization on the Illumina 850K Epic BeadChip array and processed according to kit instructions. Data were quality assessed using Illumina Genome Studio software (Illumina), and idat files were uploaded to the DKFZ Heidelberg Classifier v12.5 (www.molecularneuropathology.org).^{16 (link)}

Primer sequences for the SSR markers used for genetic map construction were described by Cai et al. [33] (link) and the sequence information of all SSR markers is provided in Table S2.
The genotyping of SNPs was performed using 6K Illumina Infinium HD Assay SNP arrays of B. napus (Illumina Inc., San Diego, CA) developed by the University of Queensland. The high-quality DNA was extracted from young leaf tissues as described by Porebski et al. [60] . Each DNA sample was diluted to a final concentration of 50 ng/ µl using ddH₂O. The SNP genotyping was carried out in accordance with the Illumina protocol (Infinium HD Assay Ultra Protocol Guide, http://www.illumina.com/).
All the SNP array data were analyzed using the Illumina GenomeStudio software (Illumina Inc., San Diego, CA), which were clustered and visualized for further analysis. Each SNP was re-checked manually to determine if any error was observed during the clustering analysis. The data processing is described by Raman et al. [39] .

Methylation levels on CpGs located in the seven YWHA genes were extracted from the methylomic data previously obtained for a subset of this cohort^{4 (link)}. Briefly, for each individual, genomic DNA (500 ng) was extracted from whole blood and treated with sodium bisulfite using the EZ-96DNA Methylation KIT (Catalog No D5004, Zymo Research, Irvine, CA, USA) following the manufacturer’s standard protocol. Genome-wide DNA methylation was assessed using Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA), which interrogates the DNA methylation profile of >485 000 CpG loci across the genome at single-nucleotide resolution. Illumina GenomeStudio software (Illumina) was used to extract signal intensities for each probe. For details about preprocessing and clean up steps, please refer to Kebir et al.^{4 (link)}. Methylation and expression data were available for 25 patients (12 converters and 13 non converters) at both baseline and at the end of follow-up. A total of 122 CpGs were located in the promoter or the body of one of the seven YWHA genes. Methylation levels on these CpG were correlated with their corresponding gene expressions using Pearson’s test.

Methylation level of patient’s DNA was quantified using the Infinium HumanMethylation27 BeadChip (Illumina, USA) according to manufacturer’s manual. Replicate samples of one subject were included as quality control for reproducibility of the assay. The Infinium BeadChip contains 27,578 CpG sites, encompassing 14,495 genes for interrogation. CpG probes with detection p-value greater than 0.05 were removed from subsequent analysis as they were not significantly different from the negative control probes and background noise. This array interrogates the methylation status at ∼97% of promoter regions defined as 2 kb around the transcription start site. Methylation values of CpG sites and their associated genomic characteristics were obtained from the Illumina® Genome Studio software (Illumina, USA). Methylation levels were reported as β-values, with a range from 0 to 1. A β-value of zero indicates a low level of methylation, while a β-value close to 1 indicates a high level of methylation. All BeadChip assays were processed at the Duke-NUS Genome Biology Facility, Singapore. Data have been deposited into the Gene Expression Omnibus (GEO) database, under the accession number GSE57956.

A total of 142 plants with stable phenotypes were selected for the DNA extraction, using a modified cetyltrimethylammonium bromide (CTAB) method and stored at −20 °C prior to array genotyping. The DNA concentration was adjusted to 50 ng/µL. We genotyped 142 F1 plants on an Illumina iScan Reader, utilizing the Infinium 8303 Potato Array (Illumina Inc., San Diego, CA). We analyzed the results using the Illumina GenomeStudio software (Illumina, San Diego, CA) [23 (link)].