A Rapid and Sensitive Next-Generation Sequencing Method to Detect RB1 Mutations Improves Care for Retinoblastoma Patients and Their Families Wenhui L.

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A Rapid and Sensitive Next-Generation Sequencing Method to Detect RB1 Mutations Improves Care for Retinoblastoma Patients and Their Families Wenhui L. Li, Jonathan Buckley, Pedro A. Sanchez-Lara, Dennis T. Maglinte, Lucy Viduetsky, Tatiana V. Tatarinova, Jennifer G. Aparicio, Jonathan W. Kim, Margaret Au, Dejerianne Ostrow, Thomas C. Lee, Maurice O'Gorman, Alexander Judkins, David Cobrinik, Timothy J. Triche The Journal of Molecular Diagnostics Volume 18, Issue 4, Pages 480-493 (July 2016) DOI: 10.1016/j.jmoldx.2016.02.006 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Coverage analysis of the RB1 next-generation sequencing test. A: The average coverage of each exon of RB1 gene, exons plus 2 bp splice junctions, the RB1 promoter, and the entire RB1 gene was plotted (from eight retinoblastoma blood samples). The line represents the coverage depth of 30×. B: Percentage of RB1 coverage on exons, the RB1 promoter, and the entire RB1 gene was plotted versus the coverage depth. The white, gray, and black bars represent the percentage of coverage on RB1 exons, the RB1 promoter, and the entire RB1 gene, respectively. The Journal of Molecular Diagnostics 2016 18, 480-493DOI: (10.1016/j.jmoldx.2016.02.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Copy number analysis of retinoblastoma samples. A: The entire RB1 gene, with vertical lines representing RB1 exons and introns between, is shown on the top. B–E: The light blue traces represent adjusted depth at all RB1 positions; the dark blue line represents the smoothed copy number (CN); and RB1 exons are labeled as four exon islands: exons 1 to 2, 3 to 6, 7 to 17, and 18 to 27. B: No copy number change in a retinoblastoma tumor sample. C: Heterozygous internal deletion of exons 2 to 6 in a retinoblastoma cell line Y79. D: Heterozygous deletion of entire RB1 gene in a retinoblastoma tumor sample. E: Homozygous deletion of exon 3 to 27 and multiplication of promoter-exon 2 of RB1 gene in a retinoblastoma tumor sample. The Journal of Molecular Diagnostics 2016 18, 480-493DOI: (10.1016/j.jmoldx.2016.02.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Evaluation of the RB1 next-generation sequencing test on mosaic mutation detection. A: Establishment of cutoffs for the mosaicism calling. Genomic DNA of two samples were mixed together with various proportions of each sample at 0%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, and 100%, respectively. Measured mosaicism rate (y axis) was plotted against its nominal rate (x axis) on the basis of the admixture proportion of two samples. Quality scores were 10, 40, 100, 200, and 400 for <10%, 11% to 25%, 26% to 50%, 51% to 75%, and >76% of measured mosaicism, respectively. B: Detection of a mosaic mutation in a retinoblastoma blood sample. The frequency of the nucleotide observed at the position (y axis) was plotted versus Phred quality score (x axis). A mosaic splice mutation c.940-2A>T was detected in the retinoblastoma sample 20. C: Detection of a mosaic mutation in a blood sample from an unaffected relative of a retinoblastoma patient. A mosaic nonsense mutation c.508G>T (p.Glu170∗) was detected in sample 21. The Journal of Molecular Diagnostics 2016 18, 480-493DOI: (10.1016/j.jmoldx.2016.02.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Quantification of MYCN copy number using the RB1 next-generation sequencing (NGS) test. A: MYCN copy number of 25 blood samples was measured with the RB1 NGS test. B: MYCN copy number of Y79 and KCN cell lines was measured. C: Admixture experiment was performed with different amounts of Y79 DNA spiked into normal control DNA of NA12878. The MYCN copy number was measured by the RB1 NGS test (solid line) as well as by the MYCN real-time quantitative PCR (qPCR) assay (broken line), and the result was plotted against the percentage of spiked-in Y79 DNA. The Journal of Molecular Diagnostics 2016 18, 480-493DOI: (10.1016/j.jmoldx.2016.02.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions