High-throughput sequencing analysis of hypermutating immunoglobulin (Ig) repertoires remains a challenging task. Scientists of Creative Biolabs has developed a robust method, we can provide the full-length profiling of human and mouse Ig repertoires with unique molecular barcoding. The achieved high-quality sequence covers the variable region of even the longest chains, along with the fragment of the constant region carrying information on the antibody isotype.
The Advent of Unique Molecular Identifiers (UMIs)
High-throughput sequencing (HTS) enables thorough investigation of the diverse immune receptors. The more thoroughly we want to analyze immune repertoires, the more complicated it becomes to quantify low-frequency clonal variants and to distinguish true homologous variants from accumulated PCR and sequencing errors. Over the past few years, advanced methods have been developed for T cell receptor (TCR) and Ig HTS profiling. There are several limitations of the current method in profiling of B cell repertoires. Firstly, it is inherent to all cDNA-based amplicon sequencing methods. It arises from substantial differences in immunoglobulin mRNA expression levels between functional subsets of B cells. Secondly, it is related to the error correction capabilities. It becomes challenging to accurately characterize B cell and T cell functional subsets, and it raises the questions of data normalization and of the influence of artificial error-based diversity on repertoire analysis. Reliable profiling of IG repertoires is especially difficult, as the initial recombinatorial diversity is further increased by the process of hypermutation. Because of this complication, until recently, deep and error-free HTS analysis of full-length IG repertoires remained practically impossible. The situation changed with UMIs, which are introduced in the process of cDNA synthesis to control bottlenecks and to eliminate errors. The use of UMIs in adaptive immunity profiling has greatly improved quantification and allowed nearly error-free analysis of immune receptor repertoires.
Fig.1 UMI-barcoded cDNA and ligation of adaptors (Turchaninova et al. 2016).
Full-length Immunoglobulin Profiling with Unique Molecular Barcoding in Creative Biolabs
Creative Biolabs developed a detailed UMI-based method for the full-length profiling of IG heavy-chain (IgH) and light-chain (IgL) repertoires. The process consists of (1) starting from cell-to-cell variation in mRNA expression levels, (2) Library preparation, (3) sequencing on our Magic™ platform, and (4) bioinformatics analysis. Such strategy provides better quantification, as the influence of cell sampling bias is decreased with the increase of sampled cell numbers, and mRNA expression levels are averaged among cells.
Equipped with world-leading technology platforms and professional scientific staff in the field of immunology, scientists of Creative Biolabs could provide both desirable depth and quality for full-length Ig profiling. We are pleased to offer the best service with the most accurate results for our global customers.
Please contact us for more information and a detailed quote.