Abstract
BackgroundNext-generation sequencing (NGS) is nowadays the most used high-throughput technology for DNA sequencing. Among others NGS enables the in-depth analysis of immune repertoires. Research in the field of T cell receptor (TCR) and immunoglobulin (IG) repertoires aids in understanding immunological diseases. A main objective is the analysis of the V(D)J recombination defining the structure and specificity of the immune repertoire. Accurate processing, evaluation and visualization of immune repertoire NGS data is important for better understanding immune responses and immunological behavior.ResultsImmunoDataAnalyzer (IMDA) is a pipeline we have developed for automatizing the analysis of immunological NGS data. IMDA unites the functionality from carefully selected immune repertoire analysis software tools and covers the whole spectrum from initial quality control up to the comparison of multiple immune repertoires. It provides methods for automated pre-processing of barcoded and UMI tagged immune repertoire NGS data, facilitates the assembly of clonotypes and calculates key figures for describing the immune repertoire. These include commonly used clonality and diversity measures, as well as indicators for V(D)J gene segment usage and between sample similarity. IMDA reports all relevant information in a compact summary containing visualizations, calculations, and sample details, all of which serve for a more detailed overview. IMDA further generates an output file including key figures for all samples, designed to serve as input for machine learning frameworks to find models for differentiating between specific traits of samples.ConclusionsIMDA constructs TCR and IG repertoire data from raw NGS reads and facilitates descriptive data analysis and comparison of immune repertoires. The IMDA workflow focus on quality control and ease of use for non-computer scientists. The provided output directly facilitates the interpretation of input data and includes information about clonality, diversity, clonotype overlap as well as similarity, and V(D)J gene segment usage. IMDA further supports the detection of sample swaps and cross-sample contamination that potentially occurred during sample preparation. In summary, IMDA reduces the effort usually required for immune repertoire data analysis by providing an automated workflow for processing raw NGS data into immune repertoires and subsequent analysis. The implementation is open-source and available on https://bioinformatics.fh-hagenberg.at/immunoanalyzer/.
Highlights
Next-generation sequencing (NGS) is nowadays the most used highthroughput technology for DNA sequencing
One way mixed lymphocyte reactions (MLRs) have been performed to identify an individuals T cells that respond against cells from one of the other potentially human leukocyte antigen system (HLA) mismatched individuals
NGS T cell receptor (TCR) β libraries were constructed for all T cell bulk samples from all five individuals and the stimulator specific T cells identified in the MLRs
Summary
Next-generation sequencing (NGS) is nowadays the most used highthroughput technology for DNA sequencing. [1, 2] This V(D)J recombination is important for the unique antigen receptors such as T cell receptors (TCR) and immunoglobulins (IG) These unique receptors and especially the third complementary-determining region (CDR3) are necessary to recognize and bind different peptides. These peptides are commonly presented by major histocompatibility complexes (MHC) and belong to potentially pathogenic microorganisms or endogenous molecules. Next-generation sequencing (NGS) is the current state of the art highthroughput technology for DNA sequencing. The advantages of this methodology, including lower costs and effort, supersede the automated Sanger method [5] in clinical and scientific research. Owing to the increased speed of DNA and RNA sequencing and continuous improvement of read length, usage of such high-throughput systems results in large amounts of data. [6]
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