Table 3 All methods implemented in IMDA (Fig. 1) for automated immune repertoire analysis

MIGEC

Read assignment by barcode (de-multiplexing) and consensus assembling based on UMIs using the open-source tool MIGEC.

I: NGS files in compressed or non-compressed FASTQ file format O: assembled reads in FASTQ file format

MiXCR

Execution of MiXCR commands for clonotype identification and quantification for receiving nucleotide and AA sequences of the CDR3 region and V(D)J gene segments.

I: files in compressed or non-compressed FASTQ file format O: immune repertoire profiling measures (e.g., V(D)J gene segments, CDRs etc.) in text file format

ContaminationAnalysis

Calculates shared UMIs and V(D)J hits of multiple samples for cross-sample contamination analysis.

I: MiXCR output, MIGEC output or non-compressed FASTQ files containing the UMI sequence in the read ID O: cleaned FASTQ files

FACSCorrection

Cell subset disambiguation of clonotypes from cells separated using FACS or magnetic sorting (e.g., CD4⁺ and CD8⁺) and elimination of clonotypes within a twofold change range.

I: filename of pairs for analysis (pairs.txt) and MiXCR output O: cleaned files in MiXCR text file format

VDJtools

Executes methods of the open-source tool VDJtools (convert and calculate diversity indices) for diversity stats visualization later-on.

I: MiXCR output O: diversity indices for all samples

Bowtie

Analyze undetermined reads from sequencing run and from MIGEC assignment using the open-source tool Bowtie2 for non-assignable read composition analysis.

I: file path to the undetermined files in compressed or non-compressed FASTQ file format and to MIGEC undef-m output file, and path to Bowtie2 library O: mapping information is collected in SAM file format

EvaluateUndetermined

If undetermined read analysis using Bowtie2 has been performed, evaluation and visualization of the results is done.

I: the Bowtie2 output in SAM file format O: read counts for each category (see Undetermined Read Processing and Analysis)

ClonalityAnalysis

Clonotype and CDR3 sequence length analysis and visualization is performed.

I: MiXCR output O: CDR3 AA length distribution and clonotype counts

DiversityAnalysis

Diversity curves are calculated and visualized as well as the diversity indices calculated using VDJtools.

I: MiXCR output and VDJtools output O: diversity curves and diversity measures

OverlapAnalysis

Shared clonotype analysis and visualization.

I: MiXCR output O: shared clonotype overlaps (heatmap and LM plots)

SimilarityAnalysis

Hierarchical clustering of all samples is performed and visualized.

I: MiXCR output O: hierarchical clustering information

VDJAnalysis

Calculation of V and J gene segment pairings and visualization using Chord diagrams.

I: MiXCR output O: chord diagrams