8.4. Alignment and clonotypes assembly using MiXCR

Clonotype extraction can be performed using MiXCR. The pipeline includes alignment of raw sequencing reads using align, error correction using correctAndSortTags, assembly of aligned sequences into clonotypes using assemble, and exporting the resulting clonotypes into tab-delimited files using exportClones. The reads can be aligned with MiXCR using the following command:

mixcr -Xmx40g analyze local:cellecta-new-singleCell-4xVBC6 \ --use-local-temp \ --export-productive-clones-only \ --tag-pattern $TAG_PATTERN \ --rigid-left-alignment-boundary \ --floating-right-alignment-boundary C \ --assemble-clonotypes-by {CDR1Begin:FR4End} \ $READ_1 \ $READ_2 \ $OUTPUT_NAME

where:

$READ_1, $READ_2 = paired end read file names (e.g. sample_a_R1.fastq.gz, sample_a_R2.fastq.gz) $OUTPUT_NAME = name of output files (e.g. sample_a)

The $TAG_PATTERN is always:

'^(R1:*)\^(MIWELL:N{10})(MIVBC:GCATCA)(R2:*)|^(MIWELL:N{10})(MIVBC:AGTCGT)(R2:*)|^(MIWELL:N{10})(MIVBC:TCGCAT)(R2:*)|^(MIWELL:N{10})(MIVBC:AGCGTA)(R2:*)'

where MIWELL specifies the well-barcode and MIVBC specifies the VBC.

The preset can be accessed by saving the file cellecta-new-singleCell-4xVBC6.yaml in the directory of the MiXCR script.

The preset cellecta-new can be accessed by saving the file cellecta-new.yaml in the directory of the MiXCR script that can be obtained here.

More details can be found on the MiXCR official website: Cellecta AIR Assay Guide.