Extended Data Fig. 6: NJ-derived neoepitopes are predicted to be processed and presented by HLA.

A-B. Density plots depicting log₂(CPM) of junction reads from RNA-seq in patient-derived GBM (A) and LGG (B) cell lines. Detectable NJ expression (colored) is validated against canonical splicing (gray). C. Read frequency spanning NJs in RPL22 and GNAS compared to the canonical junction spanning reads in glioma cell lines (n = 1). D. Density plot depicting MS analysis of publicly available LGG and GBM data sets (n = 447) reveals comparable log2(peak intensity) for NJ-derived peptides (purple) and endogenous peptides (gray). E-F. Mass spectra of peptide sequences spanning the aberrantly spliced regions in (E) RPL22 and (F) GNAS detected in publicly available glioma MS data. G. Proportion of MS-detected NJs that encode for frame-shift or in-frame mutations. H. Schematic of the selection of 192 high-confidence NJs based on RNA-seq and MS detection. I. Diagram illustrating a mechanism of neoantigen production and peptide bank generation for prediction analysis. J. Schematic depicting biological steps leading to the generation of HLA class I-presented antigens. SSNIP considers the pre-presentation steps of proteasomal processing and HLA-binding. K. Dot plot showing the overlay of the top scoring 1-percentile of HLAthena and MHCflurry 2.0 algorithms against neopeptide candidates presented by all demographically predominant HLA-A haplotypes. Top-scoring final candidates are indicated in blue as the candidates that scored in the top 1 percentile in both algorithms. L. Pie chart illustrating the distribution of neopeptide candidates found in the overlapping top 1 percentile based on composite HLA-A haplotype score. M-N. Histogram of peptide presentation likelihood scores for all top 1-percentile n-base polypeptides categorized by HLA-allele (M) or n-base polypeptides length (N), and varying n-base polypeptides lengths in HLAthena (O) and MHCflurry 2.0 (P). Q. Dot plot overlaying top 1-percentile candidates from two algorithms, highlighting final candidates (blue) scoring highly in both. j, Created in BioRender (credit: D.W.K., https://BioRender.com/r01o115; 2024).