Abstract 4485: Comparison of two commercially available T cell receptor repertoire sequencing assays

Abstract

Abstract Background: Deep sequencing of the T-cell receptor complementarity-determining regions assists with resolving T-cell diversity, and, in oncology, detects specific clones or changes in clonality associated with anti-tumor responses. However, the extreme diversity of the T-cell repertoire represents a major analytical challenge, while a gold standard method for the repertoire analysis has not yet been identified. Here, we evaluated performance of the Oncomine TCR Beta squencing assay in PBMC and formalin-fixed paraffin-embedded (FFPE) tissues, and compared it to another commercially available TCR sequencing assay. Method: RNA derived from PBMCs or tumor FFPE tissue were prepared using the Oncomine TCR Beta-SR (short read) and LR (long read) assays, and a comparator assay. Library preparation using the TCR Beta SR and LR assays required cDNA synthesis followed by TCR gene specific amplification, sample barcoding, and sequencing on the Ion Torrent S5. Comparator assay entailed gene specific cDNA synthesis, preparation of sequencing libraries, TCR gene specific amplification, sample barcoding and 2 × 150 bp sequencing on Illumina's NextSeq. Libraries generated from RNA derived from PBMCs and FFPE were sequenced to a depth of 25M and 10M reads, respectively, for the TCR Beta-SR and a comparator assay. All samples prepared with the TCR Beta-LR assay were sequenced to 3M reads. On target reads, reproducibility, sensitivity, and robustness of input were used to measure assay performance. Results: Higher fractions of on target TCR reads were identified in PBMC and FFPE libraries generated with the TCR Beta-SR and LR assays. The TCR Beta-SR and LR assays had mean pairwise correlations for replicate samples of 0.95 and 0.99, respectively, and 0.86 for a comparator assay. Rare clones at frequencies 10−5 and 10−6 in PBMC samples were detected by the Oncomine TCR Beta-SR and LR assays in samples with an input of 50 - 300 ng, where a comparator assay had inconsistent detection of the rare clones at a frequency of 10−5, independent of input. Conclusions: Thermo Fisher's Oncomine TCR Beta assay reproducibly amplifies TCR specific sequences with high sensitivity. The assay is suitable for characterization of TCR clones from RNA derived from PBMC and tissue FFPEs. Citation Format: Jennifer Mason, Martin Buchkovich, Jennifer Sims, Patrick Hurban. Comparison of two commercially available T cell receptor repertoire sequencing assays [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 4485.