Optimized IGH Chain Sequencing for Detection of Very Low Frequency B Cell Malignancies from RNA or DNA Input
Authors: Looney Timothy , Toro Michelle , Lowman Geoffrey , Chang Jayde , Pickle Loni , Topacio-Hall Denise , Hyland Fiona 
Published: November 2019

Background
High throughput sequencing (HTS) of rearranged TCRB and IGH chains has been demonstrated as a means to detect malignant T and B cells at a frequency as low as 10E-6. However, onerous input requirements (typically 20-30ug gDNA input over multiple library preparations) have impeded widespread adoption of 10E-6 as a threshold for minimal residual disease (MRD) translational research studies. Here we demonstrate an optimized highly multiplex PCR approach for amplifying IGH chains from fresh or FFPE-preserved RNA or DNA input material. Coupled with automated clonotyping and clonal lineage detection, we demonstrate detection of malignant B cell clones at a frequency of 10E-6 from a single library preparation.
Methods
Rearranged IGH chains were amplified using multiplex framework 3 and joining gene primers targeting all human IGH variable and joining gene alleles in the IMGT database (Oncomine IGH-SR assay). Libraries were generated from 25 or 100ng total RNA or 2ug gDNA derived from (1) peripheral blood leukocyte (PBL) or bone marrow (BM) spiked with Ramos B-cell cell line and (2) PBL or BM spiked with synthesized chronic lymphocytic leukemia (CLL) rearrangements from literature. Sequencing analysis was performed using the Gene Studio S5 and Ion Reporter to identify clonotypes, track clones across samples, and identify B cell clonal lineages. Clonal lineages were defined such that lineage members have a shared variable and joining gene identity, identical CDR3 lengths, and CDR3NT sequences within 85% similarity of other lineage members. Automated rarefaction analysis in Ion Reporter was used to determine optimal sequencing depth.
Results
Ramos and synthesized spike in controls were detected at a frequency of 10E-5 using 25ng of PBL total RNA and sequencing to 3M reads depth, and 10E-6 using 100ng input and 10M reads depth. gDNA-based libraries required 2ug and 3M reads depth to detect spike-in rearrangements at a frequency of 10E-5, while 10E-6 was achieved by combining the results from four 2ug gDNA libraries, each sequenced to 3M reads depth. Input and sequencing depth requirements were consistent across PBL or bone marrow derived libraries. Rarefaction analysis confirmed that the sequencing depth was appropriate for the targeted limit of detection.
Conclusions
These results demonstrate routine detection of B cell malignancy IGH chains at a frequency of 10E-6 using a limited amount of RNA or DNA material, comparing favorably to existing HTS-based approaches. We anticipate this approach to become a routine component of rare clone tracking applications including those involving B-ALL and CLL, particularly where sample material is limited or a low limit of detection is of paramount importance.

Disclosures
Looney: Thermo Fisher Scientific: Employment. Toro:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Chang:Thermo Fisher Scientific: Employment. Pickle:Thermo Fisher Scientific: Employment. Topacio-Hall:Thermo Fisher Scientific: Employment. Hyland:Thermo Fisher Scientific: Employment.