Trends in Immunology
ReviewNovel Approaches to Analyze Immunoglobulin Repertoires
Section snippets
Diversity in the Ig Repertoire
Since the seminal discovery of genetic rearrangement as the primary mechanism of Ig variability [1], immunologists have used sequence analysis to measure and interpret the vast diversity of the Ig repertoire [2]. Iterative studies aiming to estimate the number of gene segments available (see Glossary) to the recombination process 3, 4 were successfully concluded with the availability of multiple high-resolution mammalian genomes, setting the numbers used today (Table 1). Based on these variable
Conventional Bulk Sequencing
Bulk sequencing of B cell populations by NGS was first reported in 2009 [8]. The main considerations in this experimental approach center on the choice of tissue and cell population, the number of cells, and the type of nucleic acid to be used as the template (Table 2). Most workflows use cell subsets defined by flow cytometry as the starting material; however, as Ig rearrangement is restricted to B cells and only the rearranged locus can be amplified by PCR (Figure 1), a wide range of sample
Addressing the Amplification Bias
As discussed above, one central shortcoming of conventional bulk sequencing has been its inability to reliably infer the initial amount of template nucleic acid in a sample from the number of sequence reads observed for a given rearrangement. As quantification is a central aim in many repertoire analyses, various approaches have been developed to address this issue. It should, however, be noted that the problem of non-uniform Ig expression is not mitigated by any of them.
Unique Molecular Identifiers (UMIs)
UMIs are inserted
Insights
The use of Ig repertoire analysis has become especially widespread in human immunology. Here, vertical study designs face various technical and legal restrictions that limit the use of exogenous labels to track cells. In addition, they are often limited to peripheral blood as the sample material. Repertoire analysis offers a unique opportunity for cellular tracking by using Ig sequences as molecular barcodes. It can thus provide unprecedented insight by assessing not only the size and phenotype
The Unknowns of Individual Germline Diversity
The prevalent approach to the identification of unmutated V, D, and J segments is the alignment of the query sequence against a reference germline database (GLDB). Thus, the quality of the utilized GLDB is critical to data evaluation, including downstream analysis steps like clustering that depend on the identified gene segments. For an individual species, the ideal GLDB should be both complete and accurate; that is, contain all existing segments and only existing segments. Building such a GLDB
Concluding Remarks
Given the large diagnostic potential of repertoire studies, future technical development is expected to be significantly influenced by the type of additional information that can be used to leverage the plain sequencing data (e.g., cell type classification). Here microfluidic devices will gain prominence if they succeed in providing sampling depth comparable with the current methods. Single-cell approaches are likely to capitalize on their ability to provide high-dimensional, in-depth
Glossary
- Antibody-secreting cell (ASC)
- a functional classification for plasma cells and plasmablasts independent of their differentiation status.
- B cell receptor (BCR)
- the protein complex on the surface of B cells comprising a membrane-bound Ig and various signal transduction components like Igα and Igβ. Note that the exact stoichiometry of the components can vary depending on the Ig isotype.
- Birthday effect
- the fact that possible overlaps within a set of sequences scales with the square of the sequence
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