Clonal Lineage And Somatic Hypermutation Analysis Comparing Dna And Rna As Input Material By Long Amplicon Igh Chain Sequencing.

Abstract Background Traditional next generation sequencing methods for quantifying somatic hypermutation (SHM) rely on multiplex primers targeting either the framework 1 (FR1) or Leader regions of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA templates. Ion AmpliSeq primer panels for SHM evaluation were compared using both DNA and RNA input. Performance was compared using SHM values obtained from RNA samples amplified using FR1 variable gene primers in combination with constant gene primers to determine each IGH isotype and subtype in a single PCR reaction. Comparison of SHM frequencies measured from matched RNA and DNA samples were used to determine the feasibility for use of RNA in the study of SHM as well as comparison of the performance of Leader and FR1 gene primers in DNA studies. Methods Two multiplex primer panels were developed for DNA templates. These panels target the Leader or FR1 regions of the IGHV gene and the IGHJ gene. RNA samples were surveyed using FR1 and constant region primers. Comparisons of SHM frequency using 200ng of gDNA and 25ng of RNA from B cell lines and clinical research samples. Sequencing was accomplished using the Ion Gene Studio S5, while clonotyping, isotype determination, and somatic hypermutation analysis was performed using Ion Reporter 5.16 analysis software. Results Both RNA and DNA input assay workflows were able to correctly determine the SHM status of all rearrangements tested. IGHV SHM values were highly concordant between both RNA and DNA approaches. Additionally, SHM values derived from FR1 targeting variable gene primers delivered concordant results compared to Leader targeting variable gene primers when using DNA input across a wide range of SHM frequencies tested. Conclusions These results support the ability of highly multiplexed long-read NGS assays to accurately quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader targeting primers using DNA input. RNA based NGS methods benefit from lower sample requirements as well as the addition of isotype (and subtype) identification, opening new research areas for study of the B cell immune repertoire. Citation Format: Michelle Toro, Geoffrey Lowman, Loni Pickle, Stephanie Ostresh, Mark Andersen, Shrutii Sarda, Chenchen Yang. Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 511.