title: ULP-WGS and Targeted NGS in patients with Intermediate Risk Rhabdomyosarcoma: Sample PATEBP
design: Cell-free DNA, from serum, was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany) according to the manufacturers instructions. FFPE tissue was shipped to DFCI as unstained slides or scrolls and DNA was extracted using QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) with an extended lysis incubation of 3 hours to overnight. DNA was extracted from frozen tissue using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer's instructions. Germline DNA was extracted from whole blood or bone marrow using the QIAamp DNA Blood Kit (Qiagen, Germany).  Extracted DNA was quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). High-molecular-weight DNA contamination of cell-free DNA was identified by Bioanalyzer (Agilent Technologies, Santa Clara, CA) and size selection was performed for samples that demonstrated evidence of cellular DNA contamination (AMPure XP beads; Bechman Coulter, Brea, CA). Unique molecular identifiers (UMIs) were used in library preparation. Libraries were assessed for quality by Bioanalyzer followed by quantification using either the Nano Flow cell (Illumina) or the Quant-iT PicoGreen dsDNA Assay Kit.  Barcoded libraries were pooled, and ultra-low passage whole-genome sequencing was performed on an Illumina HISeq 2500 in rapid run mode or an Illumina HiSeq X to a targeted coverage of 0.1-0.2x.   For our novel custom hybrid capture panel, Rhabdo-Seq, capture baits (Twist Bioscience, San Francisco, CA) were designed specifically to detect recurrent molecular alterations associated with RMS. Libraries were pooled for custom capture and split across multiple reaction wells for hybridization to minimize batch effect variability. Multiplex pools were quantified with Quant-iT PicoGreen dsDNA Assay Kit and DNA fragment size was estimated using Bioanalyzer.   For our custom hybrid capture panel, TranSS-Seq, we normalized and pooled barcoded sequencing libraries and enriched them using the SureSelectXT Fast Target Enrichment System (Agilent Technologies) and the TranSS-Seq bait set. Postenrichment captures were sequenced with an intented coverage at target regions of 500x.