Analysis of BCR-IgH from genomic DNA with UMI incorporation

Abstract

Abstract NGS of T and B cell immune receptors has become routine in immunological studies. While using RNA as a template has its benefits, gDNA is often preferred by those collecting the sample as DNA is often easier to obtain, especially from difficult samples or samples that have been in long-term storage. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may reflect the quantity of the repertoire better. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. Here, we describe the development of a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted uniquely since the relationship of one V(D)J per cell will be maintained. Thus, both the identity and the frequency of the B-cell can be captured directly, while allowing for the elimination of PCR and sequencing error. The newly developed panel was tested on a BCR-IgH Synthetic Library. The results demonstrate a high degree of correlation with the known BCR-IgH Synthetic Library (R20.956) including coverage of all known V and J gene targets. Currently, gDNA extracted from B-cell enriched PBMC samples are being tested. This method has important implications for monitoring aberrant clonotypes in blood cancers since the frequency of a clonotype can be directly quantified, even under conditions when one clonotype is expanded.