Version Compatibility

Reference examples tested with: Ensembl VEP 111+, pVACtools 4.1+ (Frameshift plugin REPLACED the legacy Downstream in 2.0+), MHCflurry 2.1+, NetMHCpan 4.1, OptiType 1.3+ / arcasHLA, LOHHLA, WhatsHap 2.0+ (phasing), matplotlib 3.8+, numpy 1.26+, pandas 2.2+, seaborn 0.13+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Neoantigen Pipeline

"Predict neoantigens from my tumor sequencing data" -> Orchestrate HLA typing (OptiType), somatic variant calling, pVACtools neoantigen prediction, MHC binding scoring, and immunogenicity-based candidate ranking for personalized cancer immunotherapy.

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

Key Judgment -- binding is the easy part; PPV lives downstream

A binding-only pipeline has single-digit-percent positive predictive value (TESLA; Wells 2020 Cell 183:818). The critical steps are downstream of binding: correct full-resolution HLA typing (wrong allele = confident garbage), HLA loss-of-heterozygosity (run LOHHLA and DROP candidates on a lost allele; it invalidates predictions silently), proximal-variant phasing (supply --phased-proximal-variants-vcf or the mutant peptide is wrong), cancer cell fraction for clonality (clonal beats subclonal; use purity + copy number, not raw VAF), expression, and quality features (agretopicity, foreignness). Treat the ranked output as a tier-1 hypothesis list for immunopeptidomics MS and functional T-cell validation, not a final answer. Add MHC class II (CD4) neoantigens for vaccine help (see immunoinformatics/mhc-class-ii-prediction). Note on DAI below: agretopicity is most often the WT/MT binding ratio; whichever form is used, an anchor-position mutation inflates it without changing the TCR-facing surface, and a barely-presented WT makes it unstable; pair it with anchor evaluation.

Made-once commitments

| Commitment | Consequence inherited downstream | |------------|----------------------------------| | HLA typing at full 4-digit resolution (class I + II) | A wrong allele is confident garbage; every binding prediction inherits it; reconcile DNA vs RNA calls | | Variant source + somatic caller (matched-normal preferred) | Tumor-only calling leaks germline; indels/frameshifts are disproportionately valuable; expression must be RNA-confirmed and annotated INTO the VCF | | Proximal-variant phasing | Without it the mutant peptide is one the tumor never makes; germline SNPs in cis are especially treacherous | | HLA-LOH gate | Candidates on a lost allele are silently invalid (~17% pan-cancer, 30%+ HNSCC/NSCLC/cervical) |

The canonical order and why

Somatic PASS calls -> normalize + VEP-annotate (Wildtype + Frameshift plugins) -> annotate expression + DNA/RNA readcounts INTO the VCF -> PHASE proximal variants -> HLA typing + LOHHLA -> MHC binding -> clonality (CCF from purity+CN) -> quality features -> tier/rank -> pVACview review.

• Order-trap 1 - normalize + annotate with the RIGHT plugins BEFORE pVACseq. pVACseq needs the Wildtype plugin (matched WT peptide -> agretopicity) and the Frameshift plugin (novel ORF); Frameshift REPLACED the legacy Downstream in pVACtools 2.0+. Normalize before annotate.
• Order-trap 2 - PHASE proximal variants BEFORE translating the mutant peptide. THE review-sinker: editing variants independently yields a peptide the patient never makes. Merge somatic+germline, phase (WhatsHap/GATK), supply --phased-proximal-variants-vcf.
• Order-trap 3 - HLA typing (+ LOHHLA) BEFORE binding. Binding is per-allele; a wrong or lost allele makes every downstream prediction garbage. Drop LOH-lost alleles before ranking.
• Order-trap 4 - CCF/clonality from purity+copy-number BEFORE calling something subclonal. Low purity makes clonal look subclonal; correct VAF to cancer-cell fraction (copy-number/allele-specific-copy-number). Clonal beats subclonal.
• Order-trap 5 - rank WITHIN patient; do NOT hard-threshold IC50 across patients. Immunogenicity scores are relative.

Workflow Overview

Somatic VCF (annotated) + Tumor RNA-seq (optional)
        |
        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
        |
        v
[4. Immunogenicity Scoring] --> Multi-factor ranking
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        v
Ranked Vaccine Candidates (TSV + visualizations)

Prerequisites (Ensembl VEP 111+)

pip install pvactools mhcflurry vatools

mhcflurry-downloads fetch

conda install -c bioconda ensembl-vep arcas-hla 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,DQA1,DPB1,DPA1 -t 8 -o hla_output/   # type the DQA1/DPA1 alpha chains too: NetMHCIIpan needs PAIRED DQ/DP alleles

# 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:
        # arcasHLA emits 3-field alleles (A*01:01:01); pVACseq/IEDB validate 2-field (HLA-A*01:01)
        hla_alleles.append('HLA-' + ':'.join(allele.split(':')[:2]))

# 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)
# Positionals: <vcf> <expression_file> {kallisto,stringtie,cufflinks,custom} {gene,transcript}
vcf-expression-annotator somatic.vep.vcf \
    expression.tsv custom gene \
    -s tumor_sample --id-column gene_id --expression-column tpm \
    -o somatic.vep.expression.vcf

# PHASE proximal variants (the review-sinker). Merge somatic + germline, phase with WhatsHap,
# and pass the result to pVACseq via --phased-proximal-variants-vcf so a second variant in the
# same codon-window (esp. a germline SNP in cis) yields the peptide the tumor ACTUALLY makes.
whatshap phase -o phased.vcf.gz --reference reference.fa somatic_plus_germline.vcf.gz tumor.bam
tabix -p vcf phased.vcf.gz

Step 3: Run pVACseq (Ensembl VEP 111+)

# 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 \
    --phased-proximal-variants-vcf phased.vcf.gz \
    --tumor-purity 0.7 \
    --tdna-vaf 0.1 \
    --expn-val 1 \
    -t 8

Drop candidates on HLA-LOH-lost alleles (run LOHHLA/DASH) BEFORE ranking, and correct clonality to cancer-cell fraction (CCF from purity + copy number, not raw VAF; see copy-number/allele-specific-copy-number). The raw-VAF filter below is a coarse proxy.

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): WT/MT IC50 ratio (== pVACtools Fold Change), matching the
# WT/MT ratio definition. DAI > 1 = MT binds better than WT (mutation created/improved binding); higher = 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). DAI is the WT/MT IC50 ratio: >1 = MT binds better than WT.
    if row['DAI'] > 10:
        score += 2
    elif row['DAI'] > 2:
        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,DQA1*05:01-DQB1*02:01,DQA1*03:01-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,
                            include_affinity_percentile=True, verbose=0)
print(results[['peptide', 'best_allele', 'presentation_score', 'affinity', 'affinity_percentile']])

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

| Step | Parameter | Value | Rationale | |------|-----------|-------|-----------| | pVACseq | -e1 | 8,9,10,11 | MHC-I binds 8-11mer peptides | | pVACseq | -e2 | 15 | MHC-II binds 13-25mer, 15 is core | | Filtering | IC50 | <500nM | Standard strong binder threshold | | Filtering | VAF | >0.1 | Ensures clonal representation | | Filtering | Expression | >1 TPM | Detectable transcription | | Ranking | DAI (WT/MT IC50 ratio) | >2 moderate, >10 strong | MT binds better than WT (>1); higher = more tumor-specific |

Common Errors

| Symptom | Cause | Fix | |---------|-------|-----| | Peptides the tumor never makes | Proximal variants edited independently (unphased) | --phased-proximal-variants-vcf (WhatsHap/GATK); include germline in cis | | Frameshift ORFs lost / no agretopicity | Wrong/legacy VEP plugin (Downstream instead of Frameshift; missing Wildtype) | pvacseq install_vep_plugin; run --plugin Wildtype --plugin Frameshift | | Confident but invalid predictions | HLA allele wrong or on a LOH-lost haplotype | Full 4-digit typing + LOHHLA drop before ranking | | --expn-val/VAF filters silently pass everything | Expression/readcounts not annotated into the VCF | vcf-expression-annotator + vcf-readcount-annotator before pVACseq | | Clonal candidate mis-tiered subclonal | Raw VAF used as clonality on a low-purity tumor | CCF from purity + copy number (copy-number/allele-specific-copy-number) | | Candidates mis-ranked across patients | Fixed IC50 threshold applied cross-patient | Rank WITHIN patient (immunoinformatics/immunogenicity-scoring) | | No neoantigens found | Low mutation burden | Lower IC50 threshold to 1000nM; check TMB/MSI first |

References

• Hundal J, Kiwala S, McMichael J, et al (2020) pVACtools: a computational toolkit to identify and visualize cancer neoantigens. Cancer Immunology Research 8:409-420. DOI 10.1158/2326-6066.CIR-19-0401.
• Wells DK, van Buuren MM, Dang KK, et al (2020) Key parameters of tumor epitope immunogenicity revealed through a consortium approach improve neoantigen prediction (TESLA). Cell 183:818-834. DOI 10.1016/j.cell.2020.09.015. (single-digit PPV of binding-only.)
• McGranahan N, Rosenthal R, Hiley CT, et al (2017) Allele-specific HLA loss and immune escape in lung cancer evolution. Cell 171:1259-1271. DOI 10.1016/j.cell.2017.10.001. (LOHHLA.)
• Wood MA, Nguyen A, Struck AJ, et al (2020) neoepiscope improves neoepitope prediction with multivariant phasing. Bioinformatics 36:713-720. DOI 10.1093/bioinformatics/btz653. (phasing matters.)

Output Files

| File | Description | |------|-------------| | *.filtered.tsv | pVACseq filtered neoantigens | | *.all_epitopes.tsv | All predicted epitopes | | top_neoantigen_candidates.tsv | Ranked vaccine candidates | | neoantigen_summary.pdf | Visualization figures |

Related Skills

• immunoinformatics/mhc-binding-prediction - MHCflurry parameters; BA vs EL, %Rank vs nM, abundance bias
• immunoinformatics/mhc-class-ii-prediction - class II (CD4) neoantigens for vaccine help
• immunoinformatics/neoantigen-prediction - pVACtools details; LOHHLA, phasing, clonality
• immunoinformatics/immunogenicity-scoring - rank within patient (don't threshold); fitness-model quality
• immunoinformatics/epitope-prediction - B-cell epitopes
• clinical-databases/hla-typing - HLA typing (T1K is the 2024-2026 all-rounder; OptiType for class I; arcasHLA for RNA-seq); check HLA-LOH via LOHHLA / DASH which abolishes neoantigen presentation in ~17% pan-cancer (~30%+ HNSCC / NSCLC / cervical)
• clinical-databases/tumor-mutational-burden - TMB-H pan-tumor ICI biomarker; check before neoantigen-vaccine candidate selection
• clinical-databases/msi-detection - MSI-H / dMMR pan-tumor ICI biomarker; MSI-H supersedes TMB-H per Sha 2020
• clinical-databases/somatic-signatures - Clonal neoantigen burden (McGranahan 2016 Science) predicts ICI response better than total TMB
• workflows/somatic-variant-pipeline - Upstream somatic calling