Authors Ti-Cheng Chang
Publication The Neoepitope Landscape in Pediatric Cancers. Genome Medicine. 2017. 9.1: 78.
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Cancers are caused by somatically acquired alterations including single nucleotide variations (SNVs), small insertion/deletions (indels), translocations, and other types of rearrangements. The genes affected by these mutations may produce altered proteins, some of which may lead to the emergence of tumor-specific immunogenic epitopes. We developed an analytical workflow for identification of putative neoepitopes based on somatic missense mutations and gene fusions using whole genome sequencing data. The workflow has been used to characterize neoepitope landscape of 23 subtypes of pediatric cancer in the Pediatric Cancer Genome Project1.


Name Type Description Example
FastQ files (required if using FastQ inputs) Input file Gzipped FastQ files generated by experiment. Sample_R1.fastq.gz and Sample_R2.fastq.gz
BAM files (required if using BAM inputs) Input file BAM files aligned against HG19/Hg38 (WGS, WES or RNA-Seq). Sample.bam
BAM indices (required if using BAM inputs) Input file Corresponding BAM index of the BAM files above. Sample.bam.bai
Mutation file (required) Input file File describing the mutations present in the sample (special format, see below). *.txt (tab-delimited)
SNV or fusion Parameter Specify the mutation file contains SNV or gene fusion. SNV
Peptide size Parameter Size of the peptide. 9
Affinity threshold Parameter Affinity cutoff for epitope prediction report. 500

Input file configuration

Users need to provide a mutation file for SNV or gene fusion. The format of the mutation file is shown in the following example. The file can be prepared in Excel and saved as a tab-delimited text file to use as input.

The HLA alleles for testing will be derived from the HLA typing module using the workflow. The peptide size and affinity cutoff can be modified by users.

Mutation file format

GeneName Sample Chr Postion_hg19 Class AAChange mRNA_acc ReferenceAllele MutantAllele
Gene1 SampleA chr10 106150600 missense R663H NM_00101 A T
Gene2 SampleA chr2 32330151 missense N329N NM_00102 T G

Notes on preparing the above file

  • The chromosome requires a 'chr' prefix.
  • The position requires a suffix of HG19/HG38 to indicate the human genome assembly version.
  • Only the missense mutations/gene fusion is supported currently and the other types of mutations will not be processed.

Mutation file example


Name Description
Epitope affinity prediction (html) Epitope affinity. The peptide with affinity < cutoff will be highlighted.
Epitope affinity prediction (xlsx) Excel tables for the information of all epitopes
Affinity (raw output) Epitope affinity
Peptide sequence (raw output) Peptide sequences in Fasta format

Workflow Steps

HLA Typing Algorithm

The HLA typing algorithm is used to predict the HLA class I alleles. Users can either provide FastQ (paired or single end reads) or a BAM file as input. When using a BAM file as input, the reads surrounding the HLA loci and unmapped reads will be extracted. The reads will be fed into Optitype for HLA typing. The default settings for Optitype are used. The output of the HLA type can be combined with the our epitope detection algorithm to perform affinity prediction of neoepitopes.

If you use FastQ files as input:

  1. The input FastQs will be aligned against the Optitype HLA reference sequences using razers3 (see
  2. The fished FastQs will be used for HLA typing using Optitype.

If you use BAM files as input:

  1. The reads falling within the HLA loci and their paralogous loci will be extracted.
  2. The reads unmapped to the human genome will be extracted.
  3. The reads from step 1 and 2 will be combined and deduplicated (in FastQ format).
  4. The input FastQs will be aligned against the Optitype HLA reference sequences using razers3 (see
  5. The fished FastQs will be used for HLA typing using Optitype.

Epitope Prediction Algorithm

The epitope prediction algorithm first extracts peptides covering an array of tiling peptides (size defined by users) overlapping each missense mutation or gene fusion. Fusion junctions can be identified using RNA-Seq by fusion detection tools (Li et. al, unpublished). NetMHCcons3 is subsequently used to predict affinities of the peptide array for each HLA receptor in each sample. The neoepitope with affinity lower than the threshold will be highlighted in output file (default 500 nM). Below is an outline of internal steps the algorithm performs in order to generate the final report.

  1. Check the version of the genomic position of the input SNV/fusion file.
  2. Lift over the genomic coordinates if the reference genomic position is not HG19. Currently, the internal genome annotation was based on HG19 and the genome coordinates of the mutation files will be adjusted to HG19 for peptide extraction.
  3. Extract the peptide flanking the mutations.
  4. Run NetMHCcons to obtain the affinity prediction of the peptides.
  5. Produce the affinity report of each peptide.

Creating a workspace

Before you can run one of our workflows, you must first create a workspace in DNAnexus for the run. Refer to the general workflow guide to learn how to create a DNAnexus workspace for each workflow run.

You can navigate to the NeoepitopePred workflow page here.

Uploading Input files

NeoepitopePred takes the following files as input:

  • A pair of Gzipped FastQ files or an HG19/HG38 aligned BAM file. These can be generated from whole genome sequencing, whole exome sequencing, or RNA-Seq.
  • A file describing the mutations in a sample.

Refer to the general workflow guide to learn how to upload input files to the workspace you just created.

Running the Workflow

Refer to the general workflow guide to learn how to launch the workflow, hook up input files, adjust parameters, start a run, and monitor run progress.


This pipeline assumes HG19 coordinates in the mutation file. If the coordinates are based on HG38, the coordinates will lifted over to HG19 to perform epitope affinity prediction.

Analysis of Results

Each tool in St. Jude Cloud produces a visualization that makes understanding results more accessible than working with excel spreadsheet or tab delimited files. This is the primary way we recommend you work with your results.

Refer to the general workflow guide to learn how to access these visualizations.

We also include the raw output files for you to dig into if the visualization is not sufficient to answer your research question.

Refer to the general workflow guide to learn how to access raw results files.

Interpreting results

HLA typing

The output of this app contain the prediction of the HLA class I alleles from OptiType.

  • A folder stamped with the time will present in the output folder (optitype), which contains the raw output.

  • The file contains the predicted HLA alleles of the sample.

Neoepitope prediction

The output contains one summary HTML, one folder with raw output, and one folder with outputs in Excel formats:

Epitope_affinity_prediction.html (shown below):

  • This file provides a summary of the epitope prediction that can be visualized directly from web browser.
  • The peptides with affinity lower than user-defined cutoff will be highlighted in green in the webpage.

Raw_output (shown below): this folder contains the raw output of the affinity prediction. There will two major types files present here: affinity.out and flanking.seq.

affinity.out: these files are the prediction results from the netMHCcons for each peptide.

The following columns will be shown in the output:

Column Description
Gene name the name of the genes
Sample the name of the samples
Chromosome (chr) the chromosome location of the variation
Position the chromosomal position of the variation. Currently, the position will be lifted over to HG19 to ensure correct translation of peptid sequences based on the internal annotation database of the pipeline. Therefore, the position will be labeled as HG19.
Class class of the variation
Reference allele reference allele at the position
Mutant allele mutated allele at the position
mRNA_acc NCBI accession number of the mRNA
Allele HLA allele tested
Peptide the neoepitope sequences tested
Gene_variant the gene and variant residues
1-log50k Prediction score from netMHCcons
nM Affinity as IC50 values in nM
%Rank % Rank of prediction score to a set of 200.000 random natural 9mer peptides
HLAtype All of the hla alleles predicted in the specific sample

flanking.seq: these files contain the sequences used for the prediction.

XLSX: this folder contains the raw output of the affinity prediction as described above in Excel files. The files can be downloaded and opened with Excel for downstream filtering and analyses.

Frequently asked questions

None yet! If you have any questions not covered here, feel free to reach out on our contact form.

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  1. Downing JR, Wilson RK, Zhang J, et al. The Pediatric Cancer Genome Project. Nature genetics. 2012;44(6):619-622. 

  2. Szolek A, Schubert B, Mohr C, Sturm M, Feldhahn M, Kohlbacher O: OptiType: precision HLA typing from next-generation sequencing data. Bioinformatics 2014, 30:3310-3316. 

  3. Karosiene E, Lundegaard C, Lund O, Nielsen M: NetMHCcons: a consensus method for the major histocompatibility complex class I predictions. Immunogenetics 2012, 64:177-186.