A Targeted High-Throughput Next-Generation Sequencing Panel for Clinical Screening of Mutations, Gene Amplifications, and Fusions in Solid Tumors  Rajyalakshmi Luthra, Keyur P. Patel, Mark J. Routbort, Russell R. Broaddus, Jonathan Yau, Crystal Simien, Wei Chen, David Z. Hatfield, L. Jeffrey Medeiros, Rajesh R. Singh  The Journal of Molecular Diagnostics  Volume 19, Issue 2, Pages 255-264 (March 2017) DOI: 10.1016/j.jmoldx.2016.09.011 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Benefits of sequencing paired-normal controls. A: In set of 24 tumor samples, the number of variants called in the tumor samples (blue bars) is shown along with the number of bonafide somatic mutations (red bars), which were identified by comparing and filtering the germline variants called in their paired-normal control samples. The number of variant calls plotted herein are before and after applying the germline filter only to demonstrate the utility of paired-normal controls. No additional variant filters were used. B: The occurrence of variants in BRCA1/2 in 15 of the 24 patients sequenced with the paired-normal controls and the clear identification of the somatic mutations (red bars) in the background of germline variants (blue bars) is shown. The Journal of Molecular Diagnostics 2017 19, 255-264DOI: (10.1016/j.jmoldx.2016.09.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Detection of multiple genomic aberrations by OCA-Proton. Simultaneous detection of multiple copy number aberrations (CNAs) and a single-nucleotide variant in a gastrointestinal tumor with 50% tumor cellularity is shown. A: The presence of multiple CNAs is evident by the increased normalized sequencing depth of amplicons covering ESR1, FGFR1, CCND1, and MDM2 observed (red circles). The copy numbers (CNs) estimated for each gene are also shown. A PIK3CA mutation detected in the same sample is also evident in the reads, indicating the advantage of interrogating multiple variant types simultaneously by the assay. B: The approach of the panel design of sequencing both the exons and introns for genes targeted for detection of gene amplification, which facilitates better sequencing of the gene backbone, results in higher confidence of CNA detection, and is shown herein using the examples of CCND1 and MDM2 from the same sample. The sequencing reads shown herein were visualized using Integrative Genome Viewer. The Journal of Molecular Diagnostics 2017 19, 255-264DOI: (10.1016/j.jmoldx.2016.09.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Limit-of-detection (LOD) study using formalin-fixed, paraffin-embedded (FFPE) tumor samples. LOD study was performed using FFPE DNA from two tumor samples that were sequentially diluted into FFPE DNA from normal tissue. A: Sequential dilution of a tumor sample with nine single-nucleotide variants exhibited consistent detection across the dilutions, where the resultant allelic frequencies were ≥5%, with reduced detection consistency at <5%. The dotted line marking 5% allelic frequency is provided as a reference. B: LOD for gene amplifications was performed by sequencing dilutions of a tumor sample with amplification in five genes. Consistent detection of gene amplification was seen across dilutions. A dotted line marking two gene copies has been provided for reference. Both the dilutions were performed in replicates by the same technologist and sequenced on the same run. Limited variations seen in the replicates also established intrarun reproducibility of the platform. The Journal of Molecular Diagnostics 2017 19, 255-264DOI: (10.1016/j.jmoldx.2016.09.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Limit-of-detection (LOD) and reproducibility studies. The LOD for single-nucleotide variants (SNVs) and gene amplifications was established by sequencing sequentially diluted formalin-fixed, paraffin-embedded DNA samples from two cancer cell lines (DLD1 with four SNVs and SKBR3 with ERBB2 gene amplification). Sequencing of the diluted sample set was performed by two different technologists (Tech) using two different Ion Proton sequencers. High degree of detection sensitivity along with intertechnologist, interinstrument, and interrun reproducibility was observed for both SNVs and gene amplification. The dotted lines indicate 5% variant allelic frequency and normal or two gene copies, in the top and bottom panels, respectively. CN, copy number. The Journal of Molecular Diagnostics 2017 19, 255-264DOI: (10.1016/j.jmoldx.2016.09.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure 1 Summary of the various tumor and variant types tested for validation. A: A large set of 121 formalin-fixed, paraffin-embedded tumor samples and 6 cell lines was selected for validating the analytical sensitivity of the OCA-Proton platform. B: A total of 148 single-nucleotide variants (SNVs), 49 insertions or deletions (indels), 40 gene amplifications or copy number aberrations (CNAs), and 10 gene fusions were expected from the sample cohort from prior testing in the laboratory. Each of the expected mutations, gene amplifications, and fusions was detected, except four low-lying mutations (three SNVs and one indel), indicating a high level of analytical sensitivity. Neuro, neurological. The Journal of Molecular Diagnostics 2017 19, 255-264DOI: (10.1016/j.jmoldx.2016.09.011) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions