DeepNeo can predict immunogenic neoantigens in two modes. The peptides mode deals with a list of 9- or 15-mers usually containing substitution mutations whereas the protein mode identifies potential neoepitopes from a novel sequence usually derived by frameshift mutations or aberrant splicing.

[1] Peptides mode

Analyze multiple peptides as immunogenic neoepitope candidates.

1. Mutation calling with your favorite caller

Using whole exome sequencing (WES) or whole genome sequencing (WGS), perform mutation calling to obtain VCF output.

2. Extract peptide sequence around mutation

Recommended : use ensembl-vep (https://asia.ensembl.org/info/docs/tools/vep/index.html) with appropriate species and build (hg19/hg38). Make sure to download and use plugins 'Wildtype' and 'Downstream'

3. Slide to get neoantigen candidates

VEP generates lengthy protein sequences that are affected by the substitution mutation. To obtain candidates, we only need +-8 or +-14 mers neighboring the mutant amino acid.
For this process, usage of pVACtools (https://pvactools.readthedocs.io/en/latest/) - pVACseq - generate_protein_fasta command is recommended. This program will clip VEP output to get N neighboring amino acids.

4. Match HLA sequences and neoantigen candidates

With HLA typing results from your favorite program, match each HLA allele and slided neoantigen candidates of desired length (9mer for class1, and 15mer for class2 peptides) to form DeepNeo input.
*DeepNeo input : Two column tab-separated file with HLA allele and peptide.
*DeepNeo predicts HLA-A and HLA-B of MHC I, HLA-DQB1 and HLA-DRB1 of MHC II alleles. Alleles supported by DeepNeo can be found at this link.

* Different nomenclatures of HLA alleles are supported as below.
- class1 : HLA-A-0101, HLA-A01:01, HLA-A0101, HLA-A*01:01, HLA-A*0101, HLA-A-01:01, A*01:01, A0101
- class2 : HLA-DRB1-0101, HLA-DRB1*01:01, HLA-DRB1*0101, DRB1*01:01, DRB1*0101, DRB1-0101, DRB1_01_01, DRB1_0101
- mouse : H2-Db, H-2-Db, H2-IAb, H-2-IAb

5. Run DeepNeo

Enter query pMHC pairs in textbox or upload TSV file in 'Run prediction' section.
DeepNeo takes either class1 (9mer) or class2 (15mer) input, which can be specified before running the prediction. The mixture of two lengths cannot be processed; if you seek to run DeepNeo for both MHC I (9mer) and II (15mer) peptides, please run them separately.
The web page will list the first ten query pMHCs; full prediction data can be retrieved by downloading the output file.

[2] Protein mode

Analyze a protein sequence from which immunogenic neoepitopes can be identified.

1. Select MHC class

Please select corresponding MHC class. All slided peptide candidates of the desired length - 9mer for class1 and 15mer for class2 - are calculated in DeepNeo.

2. Write HLA alleles and enter a single query protein sequence

Write semi-colon separated HLA alleles. DeepNeo predicts HLA-A and HLA-B of MHC I, HLA-DQB1 and HLA-DRB1 of MHC II alleles. Alleles supported by DeepNeo can be found at this link

Enter a single query protein sequence by typing it in the text box or uploading a file in FASTA format. The peptide sequence must be within 15~100 in length; DeepNeo only takes one sequence per prediction. A description line - a line that starts with a greater-than character (">") - can be omitted.

3. Run DeepNeo

DeepNeo prediction can be performed upon clicking 'Submit' or 'Upload' button. Top ten immunogenic pMHC pairs (> 0.5 for both MHC binding and TCR reactivity prediction scores) with highest DeepNeo score (MHC binding*TCR reactivity) will be returned. The result can also be downloaded from the webpage and can be later reached using the link provided.