Analysis of human BCR-IgH from genomic DNA with UMI incorporation

Abstract

Abstract Immune repertoire analysis can be performed from practically any biological sample as long as viable nucleic acid can be extracted. While using RNA as a template has its benefits, gDNA is often preferred as gDNA is often easier to obtain, especially from difficult samples. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may better reflect the quantity of cells. 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. We developed 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 since the relationship of one V(D)J per cell will be maintained and will have a unique barcode added during the processing. The inclusion of UMI also assists with the elimination of PCR and sequencing error, which will lead to more confident analysis of low frequency clones and hypermutations. The primers were first tested on a BCR-IgH synthetic library. The sequencing result demonstrated >85% correlation with the known synthetic library gene expression. These results showed that the new gDNA BCR-IgH panel was sufficient at amplifying all known J gene targets. Presently, we have also applied this method to gDNA extracted from B cell enriched PBMC samples including the spike-in of a known BCR clone at various concentrations. To our knowledge, this is the first assay of its kind to incorporate UMI into BCR-IgH gDNA to capture both the BCR repertoire and true cell frequency.