Abstract 3305: Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing based detection of rare clonal B cells at a frequency of 10E-6
Authors: Toro Michelle , Pickle Loni , Chang Jayde , Looney Timothy , Lowman Geoffrey , Andersen Mark , Hyland Fiona 
Published: September 2020

Abstract
Background: Next generation sequencing of rearranged IGH chains has emerged as a reproducible and highly sensitive approach for detecting rare B cell clones, e.g. malignant B cell, in peripheral blood. Historically, efforts to evaluate the frequency of rare B cells by IGH chain sequencing have utilized DNA input given potential challenges in accurately quantifying template copy number from RNA data owing to B cell subtype specific variation in the expression of the B cell receptor. Hypothetically, however, RNA input based monitoring could be advantageous both owing to reduced input requirements and superior ability to detect B cell malignancies of plasmablast and plasma cell origin, where the BCR is robustly expressed. Here we compared the ability of RNA and DNA based IGH chain sequencing to detect two Burkitt's Lymphoma B cell lines (CA46 and GA10) at a frequency of 10E-6 from peripheral blood. We discuss advantages of either approach for detection of rare B cell clones.
Methods: IGH chain libraries were prepared using the Oncomine IGH-SR assay (framework 3 and joining gene multiplex primers) from gDNA or total RNA extracted from peripheral blood and spiked with Burkitt's lymphoma or CLL cell line gDNA or total RNA to a frequency of 10E-6 by mass ratio. Libraries were sequenced via the Gene Studio S5 followed by Ion Reporter analysis to identify clonotypes and evaluate B cell clone frequencies across samples. Automated downsampling analysis was used to confirm libraries were sequenced to saturation. Library preparation and analysis was performed in replicate to quantify sensitivity of detection.
Results: For each cell line, we prepared and sequenced (1) 30 libraries derived from amplification of 2ug gDNA spiked with 2pg cell line gDNA and (2) 10 libraries derived from amplification of 100ng RNA spiked with .1pg cell line total RNA. The Burkitt's lymphoma cell lines were detected in 10/30 and 8/30 gDNA libraries respectively, for CA46 and GA-10) consistent with the historical performance of orthologous DNA-based sequencing approaches. For RNA libraries, the Burkitt's lymphoma cell lines were detected in each library (10/10 and 10/10, respectively).
Conclusions: Here we demonstrate detection of B cell lines at a frequency down to 10E-6 from DNA and RNA input. Importantly, we find that RNA based IGH sequencing may significantly reduce input requirements for rare clone detection, potentially enabling routine detection of clones down to 10E-6 frequency from a single library.
Citation Format: Michelle Toro, Loni Pickle, Jayde Chang, Timothy Looney, Geoffrey Lowman, Mark Andersen, Fiona Hyland. Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing based detection of rare clonal B cells at a frequency of 10E-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3305.