Immune repertoire1/21/2024 ![]() The analytical reporting tools were integrated into the Galaxy framework ( ), as separate tools and then as ‘end to end’ workflows to utilize Galaxy’s graphical user interface (GUI). First the necessary analytical and reporting tools had to be implemented and the second was to implement a web-based application capable of use on and individual PC and on a server. To deliver a client and a server web-based immunoglobulin repertoire analysis and reporting tool required two implementation efforts. These tools provide detailed information on the rearrangement, including the specific V, D and J genes and the composition of the junctions however information extraction and visualization of these data must be completed by the user. Most frequently used tools are the ImMunoGeneTics (IMGT)/HighV-Quest, and IgBLAST. Previously, bioinformatics tools have been developed that are able to align the IGH rearrangement to the reference sequences. ![]() Analysis of IGH rearrangements is complicated since potentially all sequence reads contain a unique combination of a V, D and J gene, and stretches of nucleotides that are randomly added or deleted at the V-D and D-J junctions which is spanned by the CDR3 region (Figure 1B). To ensure accurate and reproducible determination of immunoglobulin repertoire within and between clinical laboratories thus requires standardized and reproducible data analysis. The use of next generation sequencing technology has increased the number of IgH rearrangements that can be assessed, but has also increased the amount of raw data requires technical knowledge to analyze. The immunoglobulin repertoire can be studied by sequencing the rearrangements in the immunoglobulin heavy chain (IGH) locus. IGGalaxy is available as a VM for download and use on a desktop PC or on a server. We demonstrate the use of a Galaxy virtual machine to determine the VDJ repertoire for reference data and from B-cells taken from immune deficient patients. IGGalaxy was developed for 454 NGS results but is capable of analyzing alternative NGS data (e.g. IGGalaxy provides clinical researchers with detailed insight into the repertoire of the B-cell population per individual sequenced and between control and pathogenic genomes. Re-analysis of NGS data from our samples of RAG-deficient patients demonstrated the validity and user friendliness of this tool. We demonstrate the technical validity of this platform using a standard dataset, S22, used for benchmarking the performance of antibody alignment utilities with a 99.9 % concordance with previous results. IGGalaxy supports multi-sample and multi-replicate input analysis for both igBLAST and IMGT/HIGHV-QUEST. IGGalaxy has two analysis options one using the built in igBLAST algorithm and the second using output from IMGT in either case repertoire summaries for the B-cell populations tested are available. In this study we describe ImmunoGlobulin Galaxy (IGGalaxy)- a convenient web based application for analyzing next-generation sequencing results and reporting IGH gene rearrangements for both repertoire and clonality studies. ![]() The challenge is to provide user friendly, secure and reproducible analytical services that are available for both small and large laboratories which are determining VDJ repertoire using NGS technology. Sequence analysis of immunoglobulin heavy chain (IGH) gene rearrangements and frequency analysis is a powerful tool for studying the immune repertoire, immune responses and immune dysregulation in health and disease. ![]()
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