Markus Rechsteiner, Adriana von Teichman, Jan H

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KRAS, BRAF, and TP53 Deep Sequencing for Colorectal Carcinoma Patient Diagnostics Markus Rechsteiner, Adriana von Teichman, Jan H. Rüschoff, Niklaus Fankhauser, Bernhard Pestalozzi, Peter Schraml, Achim Weber, Peter Wild, Dieter Zimmermann, Holger Moch The Journal of Molecular Diagnostics Volume 15, Issue 3, Pages 299-311 (May 2013) DOI: 10.1016/j.jmoldx.2013.02.001 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Deep sequencing workflow from tumor tissue arrival until report generation. The Journal of Molecular Diagnostics 2013 15, 299-311DOI: (10.1016/j.jmoldx.2013.02.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Quality control of PCR efficiency. PCR amplification products of nine patients with multiplex identifiers (MIDs) are shown on an agarose gel for TP53 exons 5 to 8 (A), KRAS exons 2 to 3 (B), and BRAF exon 15 (B). KRAS exon 3 from patient cr50 (double asterisks) was amplified with 5.5 and 22 ng of input DNA (C) and from patient cr47 (asterisk) with 22 ng and 263 ng of input DNA (D). In all gels, a 100-bp ladder was used. Nc, negative control; pc, positive control. The Journal of Molecular Diagnostics 2013 15, 299-311DOI: (10.1016/j.jmoldx.2013.02.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Sensitivity of deep sequencing approach. A: Total, forward, and reverse reads for the serial dilution in duplicates. B: Reads with BRAF exon 15 V600E. C: Percentage of reads with BRAF exon 15 V600E and discordant variants in duplicate. D: Number versus percentage of reads with discordant variant in duplicate. The Journal of Molecular Diagnostics 2013 15, 299-311DOI: (10.1016/j.jmoldx.2013.02.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Quality control of DNA input and MID combinations. A: KRAS exon 2 from patient cr58 was amplified with input DNA that ranged from 0.4 to 10 ng and with randomly shuffled MIDs (multiplex identifiers). B: The mutation in KRAS exon 2 G12C was detected in all amplicons by the Amplicon Variant Analyzer (AVA). C: BRAF exon 15 from patient cr88 was amplified with input DNA that ranged from 0.4 to 10 ng and with randomly shuffled MIDs. D: The mutation in BRAF exon 15 V600E was detected in all amplicons. Amplicons filtered according to our criteria described in Material and Methods are denoted by asterisks. In all gels a 100-bp ladder was used. Nc, negative control; pc, positive control. The Journal of Molecular Diagnostics 2013 15, 299-311DOI: (10.1016/j.jmoldx.2013.02.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 KRAS, BRAF, and TP53 mutations in 81 patients with colon carcinoma detected by deep sequencing. Only mutations within the coding region, with an amino acid change, and with predicted functional alteration are shown. The Journal of Molecular Diagnostics 2013 15, 299-311DOI: (10.1016/j.jmoldx.2013.02.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Validation of deep sequencing results. Two mutations just above 10% frequency are shown (A and B). One TP53 mutation was detected below 10% frequency by deep sequencing (C) and verified by an independent PCR and deep sequencing run (D). The Journal of Molecular Diagnostics 2013 15, 299-311DOI: (10.1016/j.jmoldx.2013.02.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Functional effect of TP53 mutations. A: In total, 39 nonsynonymous missense mutations, two synonymous missense mutations, six nonsense mutations, four frameshift deletions, three essential splice site mutations, and five intronic mutations were detected. B: Forty-nine mutations were grouped into driver mutations with functional effect and 10 were assumed to be neutral passenger mutations. C: The driver mutation frequencies in TP53 were scattered throughout the exons. The deletions are depicted in horizontal lines. Mutational hotspots as reported in the Cosmic database are designated with arrows. The Journal of Molecular Diagnostics 2013 15, 299-311DOI: (10.1016/j.jmoldx.2013.02.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions