Abstract 4740: Evaluation of highly-multiplexed custom panels for targeted massively parallel semiconductor sequencing on paraffin DNA
Authors: Kotoula Vassiliki , Lyberopoulou Aggeliki , Papadopoulou Kyriaki , Charalambous Elpida , Lakis Sotiris , Alexopoulou Zoi , Tsolaki Eleftheria , Lilakos Konstantinos , Fountzilas George 
Published: August 2015

Abstract
Background: Targeted massively parallel sequencing (MPS) is increasingly applied for tumor genotyping on routinely processed paraffin tissues, mostly with commercially available, predesigned multigene panels. In comparison, custom panels remain poorly addressed, despite that this approach would increase genotyping flexibility, especially in translational research in Oncology. Methods: Two custom multigene panels (B, 373 amplicons; T, 286 amplicons) were designed and evaluated with semiconductor sequencing on paired DNA sample series from 158 paraffin breast tumors and 133 matched peripheral blood samples (total n samples = 316, total n libraries = 332). The two panels shared 26.6% targeted regions (common or shifted amplicons). Amplicon performance and variant (coding and non-coding) concordance were evaluated in paired groups of samples and datasets. Results: Amplicon read ratios strongly correlated in blood and paraffin samples with Spearman's rho = 0.6055 for the B- and 0.8212 for the T-panel (both p's 75 and &lt;25, while it also differed between panels for common targets (both p's &lt;0.0001). In paired blood - tumor series, variant concordance excluding somatic variants was 92.7% with the B and 89.1% with the T panel. The highest variant concordance was observed for intra-run duplicates (99.6% for the B, and 95.8% for the T panel), with identical genotypes in 9/14 matched pairs. Genotypes obtained with different panels on the same sample were up to 81% concordant, and up to 59.9% when the same panel was applied on different samples from the same tumor. Variant frequency (VF) was constant in paraffin samples tested with the same or different panel (rho 0.959; p&lt;0.0001). High VF, coverage, and amplicon performance were significantly associated with variant concordance (all p's &lt;0.0001). However, among constantly discordant variants in paired datasets, high coverage almost exclusively corresponded to VF &lt;20%. Based on the above, genotypes for registered mutations, and hence, tumor heterogeneity were partially panel-specific. Conclusions: Highly-multiplexed custom panels with semiconductor sequencing are applicable on paraffin DNA. Efficiency and shortcomings are similar to those reported for commercially available panels. Raw data allowing for the simultaneous evaluation of amplicon performance and corresponding annotated variants would facilitate interpretation of results. The issue of panel-specific genotypes merits further investigation for all highly-multiplexed MPS applications.
Citation Format: Vassiliki Kotoula, Aggeliki Lyberopoulou, Kyriaki Papadopoulou, Elpida Charalambous, Sotiris Lakis, Zoi Alexopoulou, Eleftheria Tsolaki, Konstantinos Lilakos, George Fountzilas. Evaluation of highly-multiplexed custom panels for targeted massively parallel semiconductor sequencing on paraffin DNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4740. doi:10.1158/1538-7445.AM2015-4740