name: bio-workflows-neoantigen-pipeline description: End-to-end neoantigen discovery from somatic variants to ranked vaccine candidates. Integrates HLA typing, MHC binding prediction, pVACtools neoantigen calling, and immunogenicity scoring. Use when identifying tumor neoantigens for personalized vaccine design or checkpoint biomarkers. tool_type: mixed primary_tool: pVACtools workflow: true depends_on:

• clinical-databases/hla-typing
• immunoinformatics/mhc-binding-prediction
• immunoinformatics/neoantigen-prediction
• immunoinformatics/immunogenicity-scoring
• immunoinformatics/epitope-prediction qc_checkpoints:
• after_hla: "HLA types resolved to 4-digit, coverage adequate"
• after_binding: "Predictions generated for all alleles, IC50 <500nM filter"
• after_neoantigen: "Neoantigens identified with VAF >0.1, expressed"
• after_scoring: "Top candidates prioritized by immunogenicity" measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes. allowed-tools:
• read_file
• run_shell_command

Neoantigen Pipeline

Complete workflow from somatic variants to ranked neoantigen vaccine candidates for personalized cancer immunotherapy.

Workflow Overview

Somatic VCF (annotated) + Tumor RNA-seq (optional)
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        v
[1. HLA Typing] --> arcasHLA / OptiType (if types not provided)
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        v
[2. MHC Binding Prediction] --> MHCflurry / NetMHCpan
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        v
[3. Neoantigen Calling] --> pVACseq
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        v
[4. Immunogenicity Scoring] --> Multi-factor ranking
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        v
Ranked Vaccine Candidates (TSV + visualizations)

Prerequisites

pip install pvactools mhcflurry vatools

mhcflurry-downloads fetch

conda install -c bioconda vep arcashla optitype

Primary Path: pVACseq Pipeline

Step 1: HLA Typing (if not provided)

HLA types are critical for MHC binding prediction. If not already known from clinical testing:

# From tumor RNA-seq BAM
arcasHLA extract tumor.bam -t 8 -o hla_output/
arcasHLA genotype hla_output/tumor.extracted.1.fq.gz hla_output/tumor.extracted.2.fq.gz \
    -g A,B,C,DRB1,DQB1,DPB1 -t 8 -o hla_output/

# Parse results
cat hla_output/tumor.genotype.json

import json

with open('hla_output/tumor.genotype.json') as f:
    hla_data = json.load(f)

hla_alleles = []
for gene, alleles in hla_data.items():
    for allele in alleles:
        hla_alleles.append(f'HLA-{allele}')

# Format for pVACseq: HLA-A*02:01,HLA-A*24:02,HLA-B*07:02,...
hla_string = ','.join(hla_alleles)
print(f'HLA alleles: {hla_string}')

Step 2: VCF Annotation with VEP

pVACseq requires VEP-annotated VCF with specific fields:

# Annotate somatic VCF
vep --input_file somatic.vcf \
    --output_file somatic.vep.vcf \
    --format vcf --vcf --symbol --terms SO \
    --plugin Frameshift --plugin Wildtype \
    --offline --cache \
    --pick --fork 4

# Add expression data (optional but recommended)
vcf-expression-annotator somatic.vep.vcf \
    expression.tsv gene \
    -s tumor_sample \
    -o somatic.vep.expression.vcf

Step 3: Run pVACseq

# Basic run with MHC Class I
pvacseq run \
    somatic.vep.vcf \
    tumor_sample \
    "HLA-A*02:01,HLA-A*24:02,HLA-B*07:02,HLA-B*44:02,HLA-C*07:02,HLA-C*05:01" \
    MHCflurry MHCnuggetsI NetMHCpan \
    pvacseq_output/ \
    -e1 8,9,10,11 \
    --iedb-install-directory /path/to/iedb \
    -t 8

# With expression filtering
pvacseq run \
    somatic.vep.expression.vcf \
    tumor_sample \
    "HLA-A*02:01,HLA-A*24:02,HLA-B*07:02,HLA-B*44:02" \
    MHCflurry NetMHCpan \
    pvacseq_output/ \
    -e1 8,9,10,11 \
    --tumor-purity 0.7 \
    --trna-vaf 0.1 \
    --expn-val 1 \
    -t 8

Step 4: Filter and Rank Candidates

import pandas as pd
import numpy as np

results = pd.read_csv('pvacseq_output/MHC_Class_I/tumor_sample.filtered.tsv', sep='\t')

# Binding affinity filter (IC50 <500nM considered strong binder)
# IC50 <500nM: strong binder; 500-5000nM: weak binder
strong_binders = results[results['Median MT IC50 Score'] < 500].copy()

# Differential agretopicity index (DAI): difference between MT and WT binding
# Higher DAI = more tumor-specific
strong_binders['DAI'] = strong_binders['Median WT IC50 Score'] - strong_binders['Median MT IC50 Score']

# Expression filter (if available)
if 'Gene Expression' in strong_binders.columns:
    # TPM >1 ensures detectable expression
    strong_binders = strong_binders[strong_binders['Gene Expression'] > 1]

# VAF filter: prioritize clonal mutations
# VAF >0.1 ensures mutation present in substantial tumor fraction
strong_binders = strong_binders[strong_binders['Tumor DNA VAF'] > 0.1]

# Multi-factor scoring
def immunogenicity_score(row):
    score = 0
    # Strong binding (IC50 <150nM is very strong)
    if row['Median MT IC50 Score'] < 150:
        score += 3
    elif row['Median MT IC50 Score'] < 500:
        score += 2

    # High DAI (tumor-specificity)
    if row['DAI'] > 1000:
        score += 2
    elif row['DAI'] > 500:
        score += 1

    # Clonal mutation (high VAF)
    if row['Tumor DNA VAF'] > 0.3:
        score += 2
    elif row['Tumor DNA VAF'] > 0.15:
        score += 1

    # Expressed (if available)
    if 'Gene Expression' in row.index and row['Gene Expression'] > 10:
        score += 1

    return score

strong_binders['Immunogenicity Score'] = strong_binders.apply(immunogenicity_score, axis=1)

# Rank by composite score
ranked = strong_binders.sort_values('Immunogenicity Score', ascending=False)

# Top candidates for vaccine
top_candidates = ranked.head(20)
top_candidates.to_csv('top_neoantigen_candidates.tsv', sep='\t', index=False)

print(f'Total strong binders: {len(strong_binders)}')
print(f'Top 20 candidates exported')
print(ranked[['Gene Name', 'MT Epitope Seq', 'HLA Allele', 'Median MT IC50 Score', 'DAI', 'Immunogenicity Score']].head(10))

Step 5: MHC Class II Neoantigens (CD4+ T cell help)

pvacseq run \
    somatic.vep.vcf \
    tumor_sample \
    "DRB1*01:01,DRB1*07:01,DQB1*02:01,DQB1*03:01" \
    MHCnuggetsII NetMHCIIpan \
    pvacseq_class2_output/ \
    -e2 15 \
    --iedb-install-directory /path/to/iedb \
    -t 8

Alternative: Standalone MHCflurry

For quick binding predictions without full pVACseq pipeline:

from mhcflurry import Class1PresentationPredictor

predictor = Class1PresentationPredictor.load()

peptides = ['SIINFEKL', 'GILGFVFTL', 'NLVPMVATV']
alleles = ['HLA-A*02:01', 'HLA-B*07:02']

results = predictor.predict(peptides=peptides, alleles=alleles, verbose=0)
print(results[['peptide', 'allele', 'mhcflurry_presentation_score', 'mhcflurry_affinity']])

Visualization

import matplotlib.pyplot as plt
import seaborn as sns

fig, axes = plt.subplots(1, 3, figsize=(15, 5))

# IC50 distribution
ax1 = axes[0]
ax1.hist(ranked['Median MT IC50 Score'], bins=50, edgecolor='black')
ax1.axvline(500, color='red', linestyle='--', label='500nM threshold')
ax1.set_xlabel('Median MT IC50 (nM)')
ax1.set_ylabel('Count')
ax1.set_title('Binding Affinity Distribution')
ax1.legend()

# DAI vs IC50
ax2 = axes[1]
scatter = ax2.scatter(ranked['Median MT IC50 Score'], ranked['DAI'],
                      c=ranked['Immunogenicity Score'], cmap='viridis', alpha=0.7)
ax2.set_xlabel('MT IC50 (nM)')
ax2.set_ylabel('Differential Agretopicity Index')
ax2.set_title('Tumor Specificity vs Binding')
plt.colorbar(scatter, ax=ax2, label='Immunogenicity Score')

# Top genes
ax3 = axes[2]
gene_counts = ranked['Gene Name'].value_counts().head(15)
gene_counts.plot(kind='barh', ax=ax3)
ax3.set_xlabel('Number of Neoantigens')
ax3.set_title('Top Genes with Neoantigens')

plt.tight_layout()
plt.savefig('neoantigen_summary.pdf')

Parameter Recommendations

Troubleshooting

Output Files

*.filtered.tsv

*.all_epitopes.tsv

top_neoantigen_candidates.tsv

neoantigen_summary.pdf

Related Skills

• immunoinformatics/mhc-binding-prediction - MHCflurry parameters
• immunoinformatics/neoantigen-prediction - pVACtools details
• immunoinformatics/immunogenicity-scoring - Ranking algorithms
• immunoinformatics/epitope-prediction - B-cell epitopes
• clinical-databases/hla-typing - HLA determination methods
• workflows/somatic-variant-pipeline - Upstream somatic calling