Ffpe restoration solution

Purified DNA from breast milk specimens was sent to the University of Southern California (USC) Molecular Genomics Core
for Illumina HumanMethylation450 (HM450) BeadChip analysis. The total amount of DNA from each breast milk sample was bisulfite
treated with the Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA) and 1µl aliquots were used for MethyLight quality
control (QC) analyses to determine the completeness of conversion and the amount of converted DNA available for the HM450 assay
[26 (link)]. The remaining bisulfite converted DNA samples were further processed using the
Illumina FFPE Restoration Solution (Illumina, San Diego, CA) as specified by the manufacturer. The Restoration Solution repairs
degraded DNAs for use in genome-scale genotyping and DNA methylation assay platforms. The entire restored sample was then used as
a substrate for the Illumina HM450 BeadArrays, as recommended by the manufacturer and described previously [27 (link)]. BeadArrays were scanned using Illumina iScan readers and the raw signal intensities were extracted
from the *.IDAT files and normalized using the R package sesame [28 (link), 29 (link)], a recently developed R package that masks problematic probes (i.e. probes for which DNA
methylation is invalid because they overlap SNPs or repeats).

Cases received CLIA-certified laboratory testing for coding sequence mutations in SDHA, SDHB, SDHC, SDHD, KIT, PDGFRA, BRAF, and NF1 genes [OncoVar-GIST assay, NCI Clinical Molecular Profling Core (CMPC)]. The mean read depth for targeted gene coding sequences was >100×. Sequence reads were aligned with Burrows-Wheeler Aligner (BWA), and variants were called by mpileup and by visual inspection of alignments in Integrated Genome Viewer (IGV). Cases were assayed for genomic copy number aberrations and copy-neutral LOH with the Illumina FFPE CytoSNP assay after DNA treatment with FFPE restoration solution (Illumina) (Table 1 and fig. S2). Tumor genotyping microarray data were visualized with Nexus software (Biodiscovery Inc.). Cases were thereby annotated as SDHx-mutant versus SDHx-WT for subsequent statistical group comparisons. Additional case annotations included patient age, sex, and diagnosis of Carney triad (Table 1). The diagnosis of Carney triad was provided by treating physicians, and required clinical evidence of pulmonary chondroma in addition to GIST.