type: discovery_cohort Sex: M age: 30.1 size: 0.67 eor: 0.19 preop_growth_rate_percentyr: 0.32391094 postop_growth_rate_percentyr: 0.026787025 mnp_classifier: schwannoma mnp_score: 0.99 prior_rt: NA prior_surg: NA clinically_aggressive: 0 lf: 0 lffp: NA
Growth protocol
Patient resection specimen
Extracted molecule
genomic DNA
Extraction protocol
DNA were isolated from tumor samples using the All-Prep Universal Kit (QIAGEN, Valencia, CA)
Label
Cy5 and Cy3
Label protocol
Genomic DNA (1000 ng) is bisulfite converted using the Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA) according to the manufacturer’s recommendations. The amount of bisulfite-converted DNA as well as the completeness of bisuflite conversion for each sample are assessed using a panel of MethyLight-based real-time PCR quality control assays as described previously (PMID: 18987824). Bisulfite-converted DNA is then used as a substrate for the Illumina EPIC BeadArrays, as recommended by the manufacturer and first described in Moran, 2016 (PMID: 26673039). Specifically, each sample is whole genome amplified (WGA) and then enzymatically fragmented.
Hybridization protocol
Samples are then hybridized overnight to an 8-sample BeadArray, in which the WGA-DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. The oligomer probe designs follow the Infinium I and II chemistries, in which base extension follows hybridization to a locus-specific oligomer. With respect to the Infinium I probes, there are two different bead types for each locus, one with an oligomer that anneals specifically to the methylated version of the locus, while the other oligomer anneals to the unmethylated version of the locus. The probes terminate complementary to the interrogated CpG site for methylated loci, or complementary to the TpG for unmethylated alleles. A matched oligomer-template DNA molecule hybrid will allow for the incorporation of a labeled nucleotide immediately upstream (5’) to the interrogated CpG (or TpG) site. However, if the probe and template are mismatched, then primer extension will not occur. Adenine and thymine nucleotides are labeled with cy5 (red), while cytosine nucleotides are labeled with cy3 (green). No insertion of guanine nucleotides occurs in Infinium I assays. Of note, the identity of the dye is representative of the nucleotide adjacent to the CpG dinucleotide. The methylation discrimination is derived from separate measurements from the two different types of beads present for each locus. For some loci, both measurements will be cy3, and for others both will be cy5. The Infinium type II chemistry is a true two-color system. A matched oligomer-template DNA molecule hybrid will allow for the incorporation of a labeled nucleotide at the interrogated C or T of the CpG site. Adenine nucleotides labeled with cy5 (red) are incorporated across from unmethylated (TpG) sites, while guanine nucleotides labeled with cy3 (green) are incorporated across from methylated (CpG) sites.
Scan protocol
After the chemistry steps, BeadArrays are scanned and the raw signal intensities are extracted from the *.IDAT files using the ‘noob’ function in the minfi R package.
Description
202229250007_R02C01
Data processing
Data preprocessing and normalization was performed using minfi version 1.30 in R version 3.6.0. Methylation probes with detection significance p>0.05 were excluded from analysis. Normalization was performed using functional normalization. Probes were filtered according to the following criteria: (i) exclusion of probes mapping to sex chromosomes (n=11,551), (ii) exclusion of probes containing a common single nucleotide polymorphism (SNP) within the targeted CpG site or on an adjacent base pair (n=24,536), and (iii) exclusion of probes not mapping uniquely to the human reference genome hg19 (n=9,993). A total of 815,630 probes were retained for further analysis. Methylation β values were calculated as the ratio of methylated probe intensity to the sum of methylated and unmethylated probe intensities and visualized on heatmaps in an absolute scale.
