Tu151 - Towards Complete Haplotype Resolution: A Novel Approach for Characterizing the Immunoglobulin Heavy Chain Locus

Egil Røsjø – University of Oslo; Alan Tourancheau – L’université Paris Sciences & Lettres; Uddalok Jana – University of Louisville; Oscar Rodriguez – University of Louisville; Corey Watson – University of Louisville; Andreas Lossius – University of Oslo

Abstract Text: Immunoglobulins play a pivotal role in adaptive immune responses, and understanding their biology is crucial for unraveling mechanisms underlying infectious diseases, vaccine development, and autoimmune disorders. Despite this, the immunoglobulin heavy chain (IGH) locus has only been fully resolved in a limited number of individuals. Here, we present a novel approach utilizing Oxford Nanopore Technology (ONT) sequencing with adaptive sampling (AS) for accomplishing haplotype-resolved IGH loci.

To overcome the many complexities of the locus, we employ ultra-long sequencing to span problematic regions and large structural variants. High molecular weight gDNA from blood mononuclear cells is sequenced and enriched for the IGH locus with AS on the ONT PromethION platform, generating reads with an N50 > 60 kilobases. This enables us to bioinformatically separate the parental origin of the reads into two separate haplotypes, typically generating a single contig per haplotype with 1-3 phase blocks.

We validate this method by comparing to a well-established framework for IGH characterization, using SMRT sequencing and IGenotyper. We see comparable accuracy and enhanced contiguity using our approach on the same donors. Preliminary analysis uncovers genetic variation throughout the IGH locus, previously unreported or incompletely described. This includes a notable 150 kb segmental duplication encompassing five constant genes.

Coupled with antibody repertoire sequencing, haplotype-resolved germline data enables us to investigate recombination sites, intergenic regions, and promoters of IGH and how they might affect the adaptive immune response. This novel approach has the potential to significantly advance our understanding of the genetic underpinnings of immune response variability.