False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures Chad M. McCall, Stacy Mosier, Michele Thiess, Marija Debeljak,

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False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures Chad M. McCall, Stacy Mosier, Michele Thiess, Marija Debeljak, Aparna Pallavajjala, Katie Beierl, Kristen L. Deak, Michael B. Datto, Christopher D. Gocke, Ming-Tseh Lin, James R. Eshleman The Journal of Molecular Diagnostics Volume 16, Issue 5, Pages 541-549 (September 2014) DOI: 10.1016/j.jmoldx.2014.06.001 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Next-generation sequencing mispriming events occur at multiple amplicons within the Cancer Hotspot Panel. Representative screen capture images from the Integrative Genomics Viewer showing full-length (top of each panel) and short, misprimed reads (bottom of each panel) for EGFR exon 21 (A), RB1 exon 20 (B), APC exon 14 (C), and detail of the 5′ end of EGFR exon 21 (D). The Journal of Molecular Diagnostics 2014 16, 541-549DOI: (10.1016/j.jmoldx.2014.06.001) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Model of mispriming events during library amplification. A: The primers recognize the 5′ and 3′ ends of each amplicon (green) and have 5′ adaptors for use in emulsion PCR and sequencing (orange). B: However, in certain situations, another primer in the mix (red) is partially homologous to an internal region of the amplicon (mismatches in black). The resultant product is shorter than full length and contains false-positive mutations corresponding to the primer mismatches in the middle of the true amplicon. The Journal of Molecular Diagnostics 2014 16, 541-549DOI: (10.1016/j.jmoldx.2014.06.001) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Next-generation sequencing mispriming events correlate with low input DNA and low read depth. Modified box plots of input DNA (A) and read depth (B) versus percentage of misprimed reads at EGFR exon 21 (G873R). The horizontal line in the middle of each box indicates the median, and the top and bottom borders of the box mark the 75th and 25th percentiles, respectively. The whiskers above and below the box mark the 90th and 10th percentiles. Outliers (±1.5 × interquartile range) are represented as individual dots. ∗∗P < 0.01, ∗∗∗P < 0.001, Wilcoxon rank-sum test. The Journal of Molecular Diagnostics 2014 16, 541-549DOI: (10.1016/j.jmoldx.2014.06.001) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Model for iterative primer design for multiplex PCR reactions using NGS. Because the majority of primers do not demonstrate mispriming events, initial primers can be designed and replaced on the basis of iterative mispriming event analyses. NGS, next-generation sequencing. The Journal of Molecular Diagnostics 2014 16, 541-549DOI: (10.1016/j.jmoldx.2014.06.001) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions