Version Compatibility

Reference examples tested with: MiXCR 4.7+, VDJtools 1.2.1+, Immcantation suite 4.x, scirpy 0.24+

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

• 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.

Note: MiXCR 4.x replaced the 3.x hand-built mixcr align -s hsa -p rna-seq chain with the preset-driven mixcr analyze <preset> system, and 4.x refuses to run any command without an activated license (mixcr activate-license, or MILICENSEFILE on HPC/Docker). A copied 3.x recipe fails on both counts.

TCR/BCR Repertoire Pipeline

"Analyze my immune-repertoire sequencing data end-to-end" -> Route the data by chemistry and receptor, assemble clonotypes with MiXCR, then hand off to depth-normalized diversity (bulk TCR), clonal clustering plus somatic-hypermutation and lineages (BCR), or single-cell gene-expression integration (10x), and finally figures.

This workflow is a router, not a fixed line. Two forks decide everything downstream; pick both before running anything.

The governing principle: the pipeline forks on two axes

A repertoire measurement is a depth- and chemistry-confounded sample of an unevenly-expanded clonal population, so the correct pipeline depends on how the library was made and which receptor was sequenced. Choosing the wrong branch silently produces plausible-but-wrong numbers.

Fork A -- bulk vs single-cell

Fork B -- TCR vs BCR

The most common pipeline mistake is running BCR through exact-CDR3 VDJtools diversity. SHM shatters one clone into hundreds of near-identical variants, so exact clonotypes over-count diversity and destroy lineage structure. BCR must route to Immcantation clonal clustering (distToNearest -> findThreshold) before any diversity, SHM, or lineage step. TCR has no SHM, so exact CDR3-nt+V/J is the correct, defensible clonotype and VDJtools diversity is appropriate.

Pipeline overview

FASTQ (+ chemistry, species, receptor known)
    |
    v
[0. License + preset choice] --- mixcr activate-license ; pick preset by kit
    |
    v
[1. MiXCR analyze] ---------- <preset> R1 R2 out_prefix  ->  clones.clns + reports
    |
    +-- QC: mixcr qc / exportQc align + chainUsage
    |
    v
  FORK on data type
    |
    |-- bulk --> [2b. Export] exportClones (VDJtools) / exportAirr
    |               |
    |               v
    |            FORK on receptor
    |               |-- TCR --> [3t. DownSample to equal depth] --> CalcDiversityStats + overlap
    |               |-- BCR --> [3b. Immcantation] distToNearest->findThreshold->clones
    |               |                                -> CreateGermlines --cloned -> SHM -> Dowser trees
    |               v
    |            [4. Visualization] VDJtools / immunarch / ggplot
    |
    |-- single-cell --> [2s. exportAirr / Cell Ranger] --> [3s. scirpy]
                            chain_qc -> ir_dist -> define_clonotypes -> GEX integration
    |
    v
[5. Optional] specificity annotation (VDJdb / GLIPH2 / TCRdist) -- hypothesis, not label

Stage 0: License and preset selection

MiXCR 4.x will not run unlicensed. Activate once (academic license is free), then choose the preset by the exact kit -- the preset encodes species, RNA vs DNA, 5' boundary model (floating for multiplex primers, rigid for 5'-RACE), tag pattern, and assembling feature. The wrong preset does not error; it silently mis-calls V and truncates CDR3.

mixcr activate-license                 # or: export MI_LICENSE_FILE=/path/mi.license
mixcr exportPreset --preset-name generic-amplicon   # audit what a preset actually does

Preset by chemistry (verify against mixcr analyze --help and the built-in preset list; MiLaboratories renames occasionally):

Stage 1: MiXCR assembly (all branches)

# One command runs align -> refineTagsAndSort -> (assemblePartial) -> assemble -> export.
# From MiXCR 4.7, presets without an intrinsic assembling feature require --assemble-clonotypes-by.
# generic-amplicon REQUIRES material type + both alignment-boundary mixins (it errors without them).
# Multiplex primers on both ends -> floating boundaries; 5'RACE -> --rigid-left-alignment-boundary.
mixcr analyze generic-amplicon \
    --species hsa \
    --rna \
    --floating-left-alignment-boundary \
    --floating-right-alignment-boundary C \
    sample_R1.fastq.gz sample_R2.fastq.gz \
    results/sample

# QC every sample -- low alignment or off-target chains means wrong preset/species/contamination
mixcr qc results/sample.clns
mixcr exportQc align results/*.clns results/qc_align.pdf
mixcr exportQc chainUsage results/*.clns results/qc_chains.pdf

QC checkpoint 1 (after align): amplicon libraries should align at high rate (often >80-90%); a low rate signals wrong species, wrong boundary model, or untrimmed primers. RNA-seq mining legitimately aligns a tiny fraction -- judge it by absolute clonotype yield. chainUsage catches cross-contamination and index hopping (a TRB library showing appreciable IGH).

Detailed alignment, UMI/cell-barcode handling, and export flags: tcr-bcr-analysis/mixcr-analysis.

Stage 2: Export (branch-specific handoff)

# Bulk -> VDJtools-readable clonotype table (per chain)
mixcr exportClones -c TRB results/sample.clns results/sample.clones_TRB.tsv

# BCR or single-cell -> AIRR Rearrangement TSV (the Immcantation / scirpy interchange)
mixcr exportAirr results/sample.clns results/sample.airr.tsv

QC checkpoint 2 (after assemble): a large reads-to-clonotypes drop-off is normal (millions of reads -> thousands of clones), especially after UMI collapse. Report the right denominator: uniqueMoleculeCount on UMI libraries (reporting reads re-introduces the PCR bias the UMIs removed), cells on single-cell, reads only on non-UMI bulk.

Stage 3t: Bulk TCR diversity and overlap -- downsample FIRST

Diversity (richness, Shannon, clonality) and set-based overlap (Jaccard, shared-clonotype counts) are strictly increasing functions of sequencing depth. Comparing raw values across samples of unequal depth measures depth, not biology -- the single most common error in the field. DownSample every sample to a common depth (or read rarefaction curves at a common x) before comparing.

# 1. Equalize depth: set the target near the cohort lower quartile, and EXCLUDE (do not drag
#    everyone down to) any sample far below it -- an under-sampled library cannot support a claim.
vdjtools DownSample -x 50000 -m metadata.txt ds/

# 2. Diversity on depth-normalized samples; report the resampled table for cross-sample claims
vdjtools CalcDiversityStats -m ds/metadata.txt diversity/

# 3. Overlap with a depth-robust, abundance-weighted metric (F2 / Morisita-Horn), not Jaccard
vdjtools CalcPairwiseDistances -m ds/metadata.txt overlap/

Version caveat (MiXCR 4.x -> VDJtools): VDJtools is unmaintained for MiXCR 4.x and its parser breaks on raw exportClones output (Unable to parse clonotype string; 4.x injects commas and renames/moves columns). For a MiXCR 4.7+ cohort, prefer MiXCR's own mixcr postanalysis individual / mixcr postanalysis overlap (its native 4.x replacement for VDJtools diversity/overlap, with the same downsample-first semantics) over the vdjtools Convert -S mixcr route; if VDJtools is required, strip the added contig/target-sequence columns before Convert. immunarch (R) is the other modern alternative.

QC checkpoint 3 (before diversity): confirm all samples share one depth, and drop any sample whose rarefaction curve is still climbing steeply below that depth (under-sampled -- exclude rather than normalize the cohort down to it). Hold the clonotype match key (nt vs aa, +/-V, +/-J) constant study-wide; aa-level matching inflates apparent sharing via convergent recombination. Report clonality alongside a q=2 Hill number (inverse Simpson) and a rarefaction curve, not alone. immunarch is the modern R alternative with the same normalization semantics: tcr-bcr-analysis/vdjtools-analysis.

Stage 3b: BCR clonal clustering, SHM and lineages (Immcantation)

BCR cannot use exact clonotypes. Feed the AIRR TSV to Immcantation and follow the mandatory order: annotate -> (TIGGER genotype) -> per-sequence germline -> data-derived clonal threshold -> cluster -> per-clone germline -> SHM/selection -> lineage trees. The threshold from the bimodal distance-to-nearest distribution drives every downstream number; a wrong threshold merges or splits clones.

library(shazam); library(scoper); library(dowser)
db <- airr::read_rearrangement('results/sample.airr.tsv')
# 1. Clonal threshold: valley between the intra-clone and inter-clone modes (per dataset, never reused)
dtn <- distToNearest(db, model = 'ham', normalize = 'len')
thr <- findThreshold(dtn$dist_nearest, method = 'density')@threshold
# 2. Cluster within same-V/J/junction-length partitions at that threshold
cl <- hierarchicalClones(dtn, threshold = thr)
# 3. Reconstruct per-clone germline (CreateGermlines.py --cloned), then observedMutations, then Dowser getTrees

Cluster clones within an individual only (genotypes and thresholds are private). Diversity for BCR runs on clone_id, not exact CDR3. Full germline reconstruction, SHM quantification, BASELINe selection, and IgPhyML/Dowser trees: tcr-bcr-analysis/immcantation-analysis.

Stage 3s: Single-cell integration (scirpy)

10x paired VDJ carries native chain pairing and links receptor to cell state. The clonotype definition is a choice, not a default, and multichain cells are likely doublets.

import scirpy as ir
import mudata as mu
airr = ir.io.read_airr('results/sample.airr.tsv')           # or read_10x_vdj on Cell Ranger output
mdata = mu.MuData({'gex': gex_adata, 'airr': airr})         # scirpy 0.13+ stores AIRR as an awkward array
ir.pp.index_chains(mdata)                                    # REQUIRED before chain_qc / clonotyping
ir.tl.chain_qc(mdata)                                        # flag multichain (doublet) / orphan cells
# TCR path: exact-identity clonotypes on CDR3-nt + V/J
ir.pp.ir_dist(mdata)                                         # default metric='identity'
ir.tl.define_clonotypes(mdata)
# BCR path: SHM breaks exact identity, so cluster. ir_dist MUST be recomputed with the SAME
# metric+sequence the clustering call uses (scirpy keys the matrix as ir_dist_{sequence}_{metric};
# the identity matrix above will not satisfy a normalized_hamming call).
# ir.pp.ir_dist(mdata, metric='normalized_hamming', sequence='nt')
# ir.tl.define_clonotype_clusters(mdata, sequence='nt', metric='normalized_hamming', same_v_gene=True)
# integrate with the scanpy GEX modality; measure expansion vs cell state

Filtering multichain/orphan cells before expansion analysis preferentially deletes small clones and inflates apparent expansion -- state the trade-off, do not blindly drop them. CellRanger BCR contigs are not IMGT-numbered and include partial/nonproductive contigs; reannotate with IgBLAST (dandelion/airrflow) before rigorous BCR clustering. GEX side (clustering, annotation): single-cell/preprocessing and single-cell/clustering. Full clonotype-definition decisions: tcr-bcr-analysis/scirpy-analysis.

Stage 4: Visualization

Spectratype (CDR3-length), V-J usage circos, clonal-space bars, rarefaction curves, and clonal tracking across timepoints. Every figure inherits the depth caveat -- plot rarefaction at a common x, and track clones only after downsampling timepoints to equal depth. Recipes: tcr-bcr-analysis/repertoire-visualization.

Stage 5 (optional): Specificity annotation

Annotate or cluster clonotypes by likely antigen (VDJdb/McPAS lookup, or GLIPH2/TCRdist clustering). A database hit is a sequence match to a published antigen-specific receptor, not proof the clone binds that antigen. "Public" clonotypes are enriched for high generation-probability (Pgen), short, low-insertion CDR3s produced independently in many donors by convergent recombination (Venturi 2006 PNAS 103:18691-18696) -- publicity is not antigen selection. Treat every specificity call as a hypothesis, condition on Pgen, and validate. Handoff: tcr-bcr-analysis/specificity-annotation.

Common Errors

Related Skills

• tcr-bcr-analysis/mixcr-analysis - V(D)J alignment and clonotype assembly
• tcr-bcr-analysis/vdjtools-analysis - Depth-normalized diversity and overlap
• tcr-bcr-analysis/immcantation-analysis - BCR clonal clustering, SHM and lineages
• tcr-bcr-analysis/scirpy-analysis - Single-cell VDJ + gene-expression integration
• tcr-bcr-analysis/repertoire-visualization - Figures for the pipeline outputs
• tcr-bcr-analysis/specificity-annotation - Optional antigen-specificity annotation

References

• Bolotin DA, et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods 2015; 12:380-381.
• Shugay M, et al. VDJtools: unifying post-analysis of T cell receptor repertoires. PLoS Comput Biol 2015; 11:e1004503.
• Gupta NT, et al. Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data. Bioinformatics 2015; 31:3356-3358.
• Sturm G, et al. Scirpy: a Scanpy extension for analyzing single-cell T-cell receptor-sequencing data. Bioinformatics 2020; 36:4817-4818.
• Chao A, et al. Rarefaction and extrapolation with Hill numbers: a framework for sampling and estimation in species diversity studies. Ecol Monogr 2014; 84:45-67.
• Venturi V, et al. Sharing of T cell receptors in antigen-specific responses is driven by convergent recombination. PNAS 2006; 103:18691-18696.