A Clinically Validated Diagnostic Second-Generation Sequencing Assay for Detection of Hereditary BRCA1 and BRCA2 Mutations  Ian E. Bosdet, T. Roderick Docking, Yaron S. Butterfield, Andrew J. Mungall, Thomas Zeng, Robin J. Coope, Erika Yorida, Katie Chow, Miruna Bala, Sean S. Young, Martin Hirst, Inanc Birol, Richard A. Moore, Steven J. Jones, Marco A. Marra, Rob Holt, Aly Karsan  The Journal of Molecular Diagnostics  Volume 15, Issue 6, Pages 796-809 (November 2013) DOI: 10.1016/j.jmoldx.2013.07.004 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Distribution of sequencing reads in a single sample pool. The number of sequence reads generated for each sample in the pool 1A is expressed as the number that pass or fail the primary quality filter (prealignment) and the number that align on the target or to some off-target position within the human genome (postalignment). The horizontal lines indicate the minimum number of reads required for the lane to pass quality control. The Journal of Molecular Diagnostics 2013 15, 796-809DOI: (10.1016/j.jmoldx.2013.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Effect of library construction methods on the distribution of on-target sequence depth in sample pools. Data are from the shear-only (pool 1A, dark gray) and concatenate–shear (pool 1B, light gray) methods and show the distribution of all aligned sequences for each sample (A) and a set of data normalized by downsampling to 1 million on-target alignments per sample (B). The concatenate–shear method provides a more narrow distribution of coverage depths and a slightly higher median depth per sample. Boxplots indicate the distribution of high-quality sequence depth across the target region in each sample; the box spans the middle quartiles, the horizontal line indicates the median, whiskers indicate 1.5× the interquartile range, and circles indicate outliers. The Journal of Molecular Diagnostics 2013 15, 796-809DOI: (10.1016/j.jmoldx.2013.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Amplicon-specific differences in the distribution of sequence depth. A: The distribution of high-quality sequence depth at each base of each amplicon from a single sample (sample 59 in Supplemental Table S2). B: Boxplots indicate the distribution of the median high-quality sequence coverage for each sample in the pool 1A library; the box spans the middle quartiles, the horizontal line indicates the median, whiskers indicate 1.5× the interquartile range, and circles indicate outliers. The Journal of Molecular Diagnostics 2013 15, 796-809DOI: (10.1016/j.jmoldx.2013.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Alternate allele frequencies. For each sample pool (1A, 1B, 2A, and 2B), the ratio of high-quality reference bases to high-quality nonreference bases at every position in each sample is displayed. Known variants (blue) are found at values close to 1.0 (homozygous) or 0.5 (heterozygous), whereas nonvariant positions (green) have alternate allele ratios close to zero indicating the presence of very few nonreference bases at these positions. Known heterozygous small insertion and deletion positions (red) are found close to 0.5, as expected. A small number of positions with low-quality nonreference positions (purple) are common between multiple samples, but are correctly discarded by the variant prediction software. The Journal of Molecular Diagnostics 2013 15, 796-809DOI: (10.1016/j.jmoldx.2013.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Estimation of minimum required sequence depth. Simulations of low sequence coverage were performed by random downsampling of data from the 1A sample pool using eight final sequencing depths and 1000 replicates for all 24 patients, followed by variant prediction. A: High-quality coverage depth is plotted against on-target alignments for all nonvariant positions (range, gray band; median, solid line), all true-positive predictions (green), all false-positive predictions (blue), and all false-negative predictions (red); error bars indicate means ± SD of each estimate. Horizontal and vertical dashed lines indicate the 100-fold sequence depth and 1 × 105 on-target alignments thresholds, respectively. B: Only false-positive and false-negative predictions. C: Per-replicate count of false-positive and false-negative predictions plotted against the number of on-target alignments. False-positive and false-negative predictions are not observed at positions with greater than 100-fold coverage or when on-target alignments for the sample exceed 100,000, and these values are used for the minimum sequence data required at each position and for each sample, respectively. The Journal of Molecular Diagnostics 2013 15, 796-809DOI: (10.1016/j.jmoldx.2013.07.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions