Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor in adults, hallmarked by inter and intratumoral heterogeneity. Current treatment incorporates several biomarkers at the genomic and epigenomic levels. However, the efficacy of prognosis based on single biopsy was often undermined by the heterogeneous nature of GBM. Studies have highlighted the need for multi-sector biopsies to minimize the effect of intratumoral heterogeneity in clinical decision-making. In this project, we investigated mutations of geographically different regions of 20 primary glioblastoma specimens from seven patients for the selected regions of 13 genes using a novel targeted deep sequencing technology, Duplex Sequencing (DS). We have focused on subclonal (ultralow- and low-frequency) mutations that are not detectable by conventional next generation sequencing (NGS) methodologies but are accurately detectable by DS. Our findings indicate the heterogeneity of known GBM biomarkers, TERT promoter C228T mutation and IDH1 nonsynonymous mutations (R132H, R132G) in codon 132, in different regions of the GBM. Intratumoral heterogeneity of subclonal mutations are mainly found in EGFR, TERT, MSH6, PIK3CA, and PIK3R1 genes in most patients (six out of seven). Our results reveal that the similarity in mutation sequence context was not significantly higher in closely located specimens compared with distally located specimens. These findings could provide information on clinically relevant mutations that are unique to different regions of the tumors, and help guide future studies that seek to develop multi-sector biopsies for GBM prognosis

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