Genomic Panel Sequencing in Follicular Lymphoma: Sample CR05-3607_
DNA was extracted from 2-4 10 μm sections of each FFPE sample using the Qiagen QIAamp DNA FFPE kit (cat # 56404) according to the supplier's instructions. DNA was eluted in 40 μl ATE buffer (10 mM Tris-Cl, pH 8.3, 0.1 mM EDTA, 0.04% NaN3), quantified using a NanoDrop 2000 UV spectrophotometer (Thermo Scientific, Wilmington, DE) and assessed for quality using Qubit and gDNA trace (Qubit BR kit, Life Technologies, Darmstadt, Germany).             We designed an amplicon- (~125-175 bp) based custom exome Ion Ampli SeqTM Panel that is compatible with both MiSeq (Illumina) sequencing platform. Additional custom primers were designed to cover panel regions that were absent or displayed low coverage (<50x) with the AmpliSeq panel. Pools were amplified using the high-fidelity enzymes 5x Ion AmpliSeq HiFi Mix (pools 1 and 2) or the KAPA 2G Robust HotStart DNA Polymerase (pool 3). Immediately after amplification, PCR primers were digested using FuPa reagent (Life Technologies, Carlsbad, CA, USA) and pooled.  The concentrations and size distributions of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). After quantification, each sample-specific library was normalized to 4 nM by the addition of EB buffer (Qiagen). Samples at a concentration lower than 0.5 nM were left undiluted. Forty-eight samples per NGS MiSeq run were pooled in equal volumes, loaded onto a MiSeq V.2 300 cycle reagent cartridge and run on a MiSeq using a 2 × 150 bp paired end configuration. Libraries were sequenced at an average coverage of 5000x.
CR05-3607_