Peak pattern detection: Dynamic time warping ( Giorgino, 2009) is employed to scan for a pattern in the raw spectratypes best matching the expected Gaussian model. For sequencing data, tables containing CDR3 sequencing lengths annotated to a particular V family are used (see Supplementary Chapter S1). Preprocessing: For spectratyping raw intensity FSA files are imported, and the fluorescent peaks are matched to the expected DNA sizes of the provided size-standard ladder. Right: Boxplots of scores across all Vβ families, illustrating that GT1 reconstitutes immunodiversity better than GT2 (see also Supplementary Fig. Top: Differences in t-tests between WT versus GT1 and WT versus GT2 calculated per Vβ family. ( D) Heatmap of ImSpectR scores per sample (vertical axis) per Vβ family (horizontal axis). ( C) Vβ11 and Vβ17 peak patterns of 12 WT mice scored with ImSpectR, χ 2 Goodness-of-Fit ( Gorochov et al., 1998) and REPERTOIRE ( Long et al., 2006). ( B) CDR3 length distribution plots of Vβ11 and Vβ17 from a WT C57/Bl6 mouse and scored with ImSpectR. Residual data penalty: Area subjected to penalties (dark blue) and actual signal outside of the expected model (red). ![]() Peak Area Score: Area of the model (light blue) compared to the area of the sample (light green). Peak Height Score: Deviation of sample peaks from the overall Gaussian curve. ( A) Subscores contributing to the peak score. ImSpectR is organized in three different modules ( Fig. 1A and Supplementary Materials). Deviations from this model reflect a skewed or otherwise suboptimal immunodiversity and is used to score CDR3 length patterns (described in Supplementary Materials). Consequently, ImSpectR models the different fragment lengths of in-frame CDR3 regions with a mixture of Gaussians. The rearrangements leading to CDR3 regions are stochastic, causing the overall CDR3 length distribution in naïve T cells to adhere to a Gaussian distribution. Fragments typically display a 3 bp size difference in length, as these constitute the rearranged receptor sequences that are transcribed in-frame. Peaks in this plot represent CDR3 regions of specific sizes expressed by particular rearranged Vβ segments. A density plot of CDR3 fragment sizes ( Fig. 1A), can be used to display the junctional diversity. The distribution of these CDR3 fragment sizes represents the junctional diversity of TCRs and can thus be used to assess the diversity of the T cell repertoire. 2 Materials and methodsīriefly, amplification of CDR3 regions, either by spectratyping or sequencing, will produce fragments of different sizes (See Supplementary Chapter S0). Our package is able to assess the complexity of the distribution of CDR3 lengths from spectratype, ImmunoSeq repertoire sequencing or single cell RNA sequencing data from either human or mouse by scoring various types of deviations from the expected distribution and integrating these into one score. Here we present ImSpectR, an R package for robust scoring of immunodiversity of TCRs. As a result, CDR3 length distributions are often visually inspected, which is prone to introduce subjective variation and thus may lead to biases. Moreover, classical methods typically ignore sequences that result in out-of-frame rearrangements, which lead to non-functional T-cell receptors, and thus miss important information embedded in the complexity of the actual distribution patterns. Existing methods ( Gorochov et al., 1998 Long et al., 2006) for CDR3 length distribution analysis are typically developed to detect gross clonal abnormalities and are therefore generally not accurate enough to detect subtle changes in immunodiversity. ![]() measuring CDR3 lengths of all possible TCR Vβ-Cβ combinations by capillary electrophoresis ( Pannetier et al., 1993), is still considered to be the gold standard for clinical applications. ![]() While deep-sequencing methods have been developed for CDR3 length distribution analyses, spectratyping, i.e. To monitor immune reconstitution upon treatment, accurate quantification of the immune repertoire diversity is required. Patients with such primary immunodeficiencies often require hematopoietic stem cell transplantation or gene therapy ( Wiekmeijer et al., 2016). Indeed, rare inherited variations that hamper immune cell development generally lead to the development of severe primary immunodeficiencies, as no diverse immune repertoire can be formed. The diversity of the immune repertoire is key in combatting viral infections and other pathogens. The most diverse part of the receptor is the third complementarity-determining region 3 (CDR3) that binds a specific antigen. The immune repertoire refers to the diversity of T-cell receptors (TCR) and B cell receptors (BCR) on T- and B-lymphocytes each expressing a unique antigen-specific receptor.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |