Assemble full TCR/Ig receptor sequences

Tip

MiXCR provides analyze command that packs a complicated execution pipelines (alignment, assembly, exporting etc.) into a single command. We recommend to use analyze for most types of input libraries instead of manual execution of all MiXCR analysis steps. To assemble full TCR/IG receptor sequences with analyze command, one should simply use --contig-assembly option of analyze.

MiXCR allows to assemble full TCR/Ig receptor sequences (that is all available off-CDR3 regions) with the use of assembleContigs command. Full sequence assembly may be performed after building of initial alignments and assembly of ordinary CDR3-based clonotypes. The typical workflow for full receptor assembly of e.g. mouse B-cells may be the following:

# align raw sequences
mixcr align --species mmu -p kAligner2 --report report.txt input_R1.fq input_R2.fq alignments.vdjca

# assemble default CDR3 clonotypes (note: --write-alignments is required for further contig assembly)
mixcr assemble --write-alignments --report report.txt alignments.vdjca clones.clna

# assemble full BCR receptors
mixcr assembleContigs --report report.txt clones.clna full_clones.clns

# export full BCR receptors
mixcr exportClones -c IG -p fullImputed full_clones.clns full_clones.txt

Note that at assembly stage we specified --write-alignments option that enables .clna file format for storing clones and alignments to clones mapping. This mapping is used then by the assembleContig algorithms. The output of assembleContig is a standard binary file with clonotypes (.clns). To export the full information about assembled full IG receptor sequences it is recommended to use the option -p fullImputed in exportClones. With this option the germline nucleotide sequences will be used for uncovered regions of gene features (marked lowercase). The output will look like:

cloneId cloneFraction aaSeqImputedFR1 aaSeqCDR3
0 0.061 qvqlqqwgagllkpsetlslTCAVY CARKKLEGRFDYW
1 0.054 qvqlvesgggvvqpgrslrlscaAS CARQGQA_*RQVDPW

To print help for assembleContigs use:

mixcr help assembleContigs

Full sequence assembler parameters

To pass specific option for the full sequence assembler use the following syntax:

mixcr assembleContigs -Oparameter=value input.clna output.clns

The following options are available:

Parameter Default value Description
subCloningRegion CDR3 Region where variants are allowed
minimalContigLength 20 Minimal contiguous sequence length
alignedRegionsOnly false Assemble only parts of sequences covered by alignments
branchingMinimalQualityShare 0.1 Minimal quality fraction (variant may be marked significant if variantQuality > totalSumQuality * branchingMinimalQualityShare
branchingMinimalSumQuality 80 Minimal variant quality threshold (variant may be marked significant if variantQuality > branchingMinimalSumQuality
decisiveBranchingSumQualityThreshold 120 Variant quality that guaranties that variant will be marked significant (even if other criteria are not satisfied)
outputMinimalQualityShare 0.5 Positions having quality share less then this value, will not be represented in the output
outputMinimalSumQuality 50 Positions having sum quality less then this value, will not be represented in the output
alignedSequenceEdgeDelta 3 Maximal number of not aligned nucleotides at the edge of sequence so that sequence is still considered aligned “to the end”
alignmentEdgeRegionSize 7 Number of nucleotides at the edges of alignments (with almost fully aligned seq2) that are “not trusted”
minimalNonEdgePointsFraction 0.25 Minimal fraction of non edge points in variant that must be reached to consider the variant significant