Simultaneous Detection of B Cell Receptor Rearrangements and Joining Gene Translocations Via Ring-Seq, a Novel Multiplex PCR Technology for Targeted Next-Generation Sequencing of Translocations with Novel Gene Partners or Breakpoints

Introduction Next-generation sequencing (NGS) of B cell receptor rearrangements is a leading method for clonality assessment and measurable residual disease (MRD) monitoring of B cell malignancies. Current methods rely upon multiplex PCR (mPCR) amplification of rearranged IGH, IGK or IGL loci via variable and joining gene targeting primers followed by NGS of amplicons to assess clonality and determine the presence of marker rearrangements. Limitations of this approach include a need for reflex testing owing to somatic hypermutation (SHM) mediated loss of sensitivity and an inability to detect clinically relevant translocations involving immune receptor loci. Here we introduce Ring-Seq, a novel approach for the targeted detection of gene fusions, including those with unknown partners or breakpoints. Unlike traditional mPCR immune repertoire assays, the Ring-Seq protocol leverages highly efficient template circularization coupled with inverse PCR to enable targeted sequencing of VDJ rearrangements and translocations while avoiding loss of sensitivity due to variable gene SHM. Using the Singular Genomics G4TM sequencing platform, we apply Ring-Seq to simultaneously identify clinically relevant translocations and VDJ rearrangements of the IGH locus from highly degraded material. Methods The Ring-Seq method begins with a highly efficient intramolecular ligation of DNA fragments followed by a multiplex inverse PCR of joining genes that preferentially amplifies breakpoint junction containing fragments. Amplicon libraries are sequenced on the G4 platform via 2x150bp reads and analyzed to detect translocations and immune receptor rearrangements. We pooled gDNA from reference cell lines known to harbor marker IGH VDJ rearrangements (Ramos, CA-46) and BCL1-JH and BCL2-JH translocations (IVS-0010 and IVS-0030, Invivoscribe Cat #40880550 and 40881750 respectively). Pooled reference gDNA was spiked into a background of healthy donor peripheral blood leukocyte (PBL) gDNA to evaluate performance over a range of marker frequencies. To simulate performance with degraded materials, gDNA input was fragmented to ~200bp length by sonication (Covaris) prior to analysis. Results BCL1-JH and BCL2-JH translocations and marker IGH VDJ rearrangements were detected from 50ng pooled reference control gDNA. Translocations and marker VDJ rearrangements were also detected in 50ng samples consisting of fragmented reference control material spiked at 1% frequency into a background of fragmented healthy donor PBL. We observed a high on-target rate, enabling marker detection with less than 1M reads per sample in all conditions tested. Conclusions We have applied a novel technology for multiplex inverse PCR, Ring-Seq, to enable the simultaneous detection of B cell receptor rearrangements and clinically relevant JH translocations, including those involving non-canonical breakpoint regions. Unlike traditional mPCR approaches for B cell receptor sequencing, Ring-Seq avoids targeting the highly mutable variable gene region, thereby obviating the need for reflex testing due to SHM-mediated loss of sensitivity. We expect the approach to be particularly well suited to the future identification and monitoring of translocation-associated B cell malignancies such as mantle cell lymphoma.