Classification of pediatric soft and bone sarcomas using DNA methylation-based profiling
Pediatric sarcomas present heterogeneous morphology, genetics and clinical behavior posing a challenge for an accurate diagnosis. DNA methylation is an epigenetic modification that coordinates chromatin structure and regulates gene expression, determining cell type and function. DNA methylation-based tumor profiling classifier for sarcomas (known as sarcoma classifier) from the German Cancer Research Center (Deutsches Krebsforschungszentrum) was applied to 122 pediatric sarcomas referred to a reference pediatric oncology hospital. The classifiers reported 88.5% of agreement between histopathological and molecular classification confirming the initial diagnosis of all osteosarcomas and Ewing sarcomas. Transcriptome raw data quality was verified with FASTQC. We used STAR-fusion to identify and annotate fusion transcripts based on discordant read alignments with default configurations. ChimeraViz was used to plot fusion-genes. Overall design: RNA was extracted with RNeasy Kit (QIAGEN, Hilden, Germany; cat. ID: 74106) and quantified with High Sensitivity Qubit™ RNA Assay Kits (Invitrogen™, Waltham, MA, USA; cat. ID: Q32855). Transcriptome assay was performed using Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (Illumina, San Diego, CA, USA) and sequenced on the NextSeq550 or NOVAseqX (Illumina Inc, San Diego, CA, USA). Paired-end runs were sequenced in 74 cycles per read (2 X 74), with an average of 41.5 million reads per sample.