Computational protocol: Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens

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Protocol publication

[…] The HLA peptides were dried by vacuum centrifugation, re-solubilized with 0.1% Formic acid and resolved on capillary reversed phase chromatography on 0.075×200 mm laser-pulled capillaries, self-packed with 3μ Reprosil-Aqua C18 []. Electrospray tandem mass spectrometry was performed with the Q-Exactive-Plus mass spectrometer (Thermo Scientific). The MS data was analyzed by MaxQuant [] version 1.4.1.2, with 5% FDR and by Sequest using the Proteome Discoverer version 1.4.1.14 (Thermo Scientific). Peptide identifications were based on the human section of the Uniprot database (http://www.uniprot.org) from February 2014 combined with the wild type and mutant protein sequences. [...] RNA was extracted from 12T cells using RNeasy mini kit (Qiagen). 1 μg of total RNA was processed using the TruSeq RNA Sample Preparation Kit v2 protocol (Illumina). Libraries were evaluated by Qubit and TapeStation. Sequencing libraries were constructed with barcodes to allow multiplexing of few samples on one lane of Illumina HiSeq 2500 V4 instrument. About 60 million single-end 60-bp reads were sequenced.The TopHat (v2.0.10) was used to align the reads to the human genome (hg19), and counting reads on hg19 refseq genes (downloaded from igenomes) was done with HTSeq-count (version 0.6.1p1).RPKM values were calculated using Total exonic length for each gene, which was calculated using the bioconductor GenomicFeatures package. […]

Pipeline specifications

Software tools TopHat, HTSeq, GenomicFeatures
Databases iGenomes
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Melanoma, Neoplasms
Chemicals Amino Acids