Extraskeletal myxoid chondrosarcoma pelvic metastasis
Sarcoma tissue and PBMC were collected fresh from the operating room and processed immediately according to Pitt Biospecimen Core and Musculoskeletal Oncology Lab protocols. DNA was extracted from the frozen samples using the QIAamp Fast DNA Tissue Kit (Qiagen, Cat#51404). DNA concentration was determined with the Qubit 3.0 Fluorometer and DNA Broad Range Kit (ThermoFisher, Cat#Q32580). Samples were then submitted to the University of Pittsburgh Medical Center (UPMC) Genome Center (UGC) for further preparation, sequencing, and analysis. The samples were processed following standard WGS pipeline in UPMC UGC. Extracted DNA was quantitated using Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific). DNA libraries were prepared using the KAPA Hyper Plus Kit (Roche). 500 ng of genomic DNA was processed through fragmentation, enzymatic end-repair and A-tailing, ligation, followed by quality check using Fragment Analyzer (Agilent). The library size assessment was done using Fragment Analyzer (Agilent). Libraries with an average size of 400 bp (range: 200-600bp) were quantified by qPCR on the LightCycler 480 (Roche) using the KAPA qPCR quantification kit (Roche). The libraries were normalized and pooled by calculating the nM concentration based on the fragment size (base pairs) and the concentration (ng/mL) of the libraries by qPCR result. Sequencing was performed using NovaSeq 6000 platform (Illumina) with 151 paired-end reads to an average target depth of 30-50X for germline and 87X, 74X, and 115X coverage for P, LM, and PM tumor samples respectively. Concatenated base call files generated on NovaSeq 6000 platform were converted to sample level FASTQ using Illumina's bcltofastq v2.20. The quality of paired FASTQ reads was checked using FASTQC v0.11.3-1. The paired FASTQ reads were mapped to GRCh38 reference genome hs38DH (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155401), duplicates were marked, and germline variants were called using Sentieon DNAseq pipeline v2.0.1-201911.012 (Kendig et al., 2019). Picard tools v2.4.1(Broad Institute, 2016) were run on alignment BAM to provide mapping quality and metrics. Zou T, Sethi R, Wang J, Budak G, John I, Chandran U, et al. Whole Genome Sequencing for Metastatic Mutational Burden in Extraskeletal Myxoid Chondrosarcoma. Frontiers in Molecular Medicine. 2023 Mar 28.