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TMB was calculated because the ratio between the variety of mutations and the size of the sequenced regions (35.7 Mb). For these sufferers with available RNAseq, quartiles of FPKM were calculated. Mutations identified by WES analysis were thought-about as “expressed mutations” if no less than one of the gene counter algorithms was expressed in 3rd or 4th quartile. Once mutations have been filtered, 29-mer amino acid sequences, containing the mutated residue at position 15, have been designed. These sequences have been applied to NetMHCPan 4.zero to foretell peptide binding to patients’ own HLA class I molecules.
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Duplicates had been marked and Sorted utilizing Picard’s MarkDuplicates device. Reads have been then cut up and trimmed utilizing GATK SplitNTrim device. In/Del realignment and base recalibration had been carried out using GATK toolbox. Gene counts have been calculated utilizing featureCount , Cufflinks and HTseq .
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Plat-A cells (8×105 cells/well) had been seeded 24h earlier than transfection in 6-well plates in 2 mL/well of infection medium (Plat-A medium with out antibiotics). 20-24h later, once the cells had reached 70% confluence, 500μL/well of a mix containing plasmids and Lipofectamine had been added. The mixtures contained 3 μg/well of transgene carrying plasmid, 2 μg/well of pMD2.G and 10 μL/well of Lipofectamine 2000, and have been prepared in OPTIMEM medium in accordance with the manufacturer´s protocol. PMD2.G was a gift from Didier Trono (Addgene plasmid # 12259). The supernatant containing the retroviruses was collected 48h and 72h post-transfection. Debris was eliminated by centrifugation at 2000rpm for 1 minute.
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In our case, along with a binding threshold for all mutated peptides, we distinguished two groups concerning the binding capability of the WT peptide. In the first group , where WT peptides were poor or null binders, an essential proportion of peptides contained the mutation at HLA-anchoring residues (e.g. peptides CAM69-6, CAM73-9). However, in the second group all peptides contained the mutation at non-anchor residues.
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