Genome-wide sequencing in glioma samples provides comprehensive insights into oncogenesis and malignant transformation. Several distinct biomarkers have been proven to have clinical significance and are being widely applied in routine clinical practice. Standard sample processing lays the foundation for successful molecular testing. In this study, we found intraoperative neuronavigation ensured higher tumor purity during sample collection, and an automated device helped improve DNA quality and increased yields. These two technologies are beneficial for glioma tissue bank construction and provide for accurate molecular testing during routine clinical practice.