NeoantigenLab Protocol Pack — 6 Step-by-Step Protocols for Neoantigen Research
6 step-by-step protocols for the full neoantigen pipeline — from raw WES data to clinic-ready candidates.This pack is written for computational biologists and wet-lab researchers who want clear, reproducible protocols without wading through scattered documentation.---### What's includedProtocol 1 — End-to-end neoantigen prediction (WES → pVACseq)Full pipeline: variant calling with GATK Mutect2, VEP annotation, pVACseq execution, and output interpretation. Includes exact commands, a parameter reference table, and guidance on filtering candidates for vaccine vs. TIL applications.Protocol 2 — HLA typing: OptiType, HLA-HD, and POLYSOLVERWhen to use each tool, exact commands, how to validate calls with LOHHLA, and how HLA LOH affects which alleles to include in pVACseq.Protocol 3 — TIL therapy: tumor resection to patient infusionWeek-by-week protocol covering tumor processing, Pre-REP expansion, REP (G-Rex protocol), QC release criteria, and lymphodepletion. Includes the checkpoints used in the lifileucel (Amtagvi) manufacturing playbook.Protocol 4 — mRNA neoantigen vaccine designNeoantigen selection criteria, 25-mer peptide design, codon optimization, UTR design, concatenation order optimization, and junction neoantigen filtering — based on the mRNA-4157/V940 (Moderna/Merck) design framework.Protocol 5 — Tumor mutational burden (TMB) calculationExact calculation from WES VCF, germline filtering with gnomAD, clinical interpretation table, and the FDA companion diagnostic cutoff context (KEYNOTE-158).Protocol 6 — Checkpoint inhibitor selection by biomarkerBiomarker priority framework (MSI-H → TMB-H → PD-L1 TPS/CPS by indication), 2026 drug reference table covering pembrolizumab, nivolumab, atezolizumab, ipilimumab, and relatlimab+nivolumab, and clinical interpretation guidance.Plus: Quick-reference tool comparison tables (HLA typing, binding predictors, databases) and a troubleshooting guide for 8 common pipeline errors.Fact-checked: Every command, flag, and clinical cutoff has been checked against current tool documentation and literature (pVACtools, OptiType/HLA-HD/POLYSOLVER docs, FDA labels) — including corrections most guides get wrong (pVACseq's actual CLI flags, TIL eligibility criteria, PD-L1 TPS vs. CPS by indication).---### Who this is for- Computational biology researchers running neoantigen pipelines- Wet-lab immunologists starting TIL therapy programs- Clinical researchers designing neoantigen vaccine trials- Anyone who has read the NeoantigenLab blog posts and wants the protocols behind them---### Format- Single PDF, 16 pages- Exact shell commands throughout- Parameter tables with recommended values and clinical cutoffs- No filler — written to be used as a bench reference---### From the blogThis pack is the protocol companion to the NeoantigenLab blog series at neoantigenlab.com, covering pVACseq, HLA typing, TIL therapy, mRNA vaccines, TMB, and checkpoint inhibitors.
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