Whole-genome sequencing of FFPE tumors specimens from pediatric patients.
Surgical samples were placed into standard tissue cassette(s) and completely immersed in fresh 10% Neutral Buffered Formalin (NBF) and loaded into the Peloris tissue processor. Surgical routine samples were processed for about 7 hours in multiple steps per reagent consisting of formalin (90 minutes), alcohol (125 minutes), xylene (90 minutes) and paraffin (90 minutes). Cassettes were then removed and embedded in paraffin blocks using the Tissue-Tek AutoTEC a120. FFPE tissue was deparaffinized using heat treatment (90°C for 10' in 480μL PBS and 20μL 10% Tween 20), centrifugation (10,000xg for 15') and ice chill. Paraffin and supernatant were removed, and the pellet was washed with 1mL of 100% EtOH followed by an incubation overnight in 400µl of 1M NaSCN for rehydration and impurity removal. Tissues were subsequently digested with 40µl of Proteinase K (600 mAU/ml) in 360µl Buffer ATL at 55°C. DNA isolation proceeded with the Mag-Bind Blood & Tissue DNA HDQ 96 Kit (Chemagic SKU M6399-01) according to the manufacturer's protocol modified by replacing AW2 buffer with 80% ethanol. All DNA was eluted in 0.5X Buffer AE. 500 ng of genomic DNA were sheared where possible for all but 5 samples (minimum gDNA: 140.9ng) using a LE220-plus Focused-ultrasonicator (Covaris catalog # 500569). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) and were subjected to a 0.5X size select using aMPure XP beads (Beckman Coulter catalog # A63882) after post-ligation cleanup. 5 cycles of PCR were performed.