Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients Katharina König, PhD, Martin Peifer,

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Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients Katharina König, PhD, Martin Peifer, Jana Fassunke, PhD, Michaela A. Ihle, Dipl-Biol, Helen Künstlinger, Dpil-Biol, Carina Heydt, MSc, Katrin Stamm, PhD, Frank Ueckeroth, Claudia Vollbrecht, BSc, Marc Bos, MD, Masyar Gardizi, MD, Matthias Scheffler, MD, Lucia Nogova, MD, Frauke Leenders, MD, Kerstin Albus, MSc, Lydia Meder, MSc, Kerstin Becker, PhD, Alexandra Florin, MA, Ursula Rommerscheidt-Fuss, Janine Altmüller, PhD, Michael Kloth, MD, Peter Nürnberg, Prof, Thomas Henkel, PhD, Sven-Ernö Bikár, PhD, Martin L. Sos, PhD, William J. Geese, PhD, Lewis Strauss, Yon-Dschun Ko, MD, Ulrich Gerigk, MD, Margarete Odenthal, PhD, Thomas Zander, MD, Jürgen Wolf, Prof, Sabine Merkelbach-Bruse, MD, Reinhard Buettner, MD, Lukas C. Heukamp, PhD, MD Journal of Thoracic Oncology Volume 10, Issue 7, Pages 1049-1057 (July 2015) DOI: 10.1097/JTO.0000000000000570 Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Schematic presentation of the library preparation. A and C, Genomic DNA (up to 50 ng) is amplified with the AmpliSeq Custom Panels. Primers are digested with FuPa, an “A” is added at the 3´ end to allow adapter ligation. After bead clean up and size selection, the successfully ligated products are amplified by PCR with a final bead clean up. Samples are quantified by Qubit, normalized to 10 nM, and pooled to be then sequenced on v2 MiSeq Cartridge. A table outline (A) and a cartoon (C) are depicted. B, Mutational analysis can be completed within 4 working days starting with the DNA extraction until data analysis. PCR, polymerase chain reaction. Journal of Thoracic Oncology 2015 10, 1049-1057DOI: (10.1097/JTO.0000000000000570) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Discrepancies between SS and NGS. A–C, Three of 11 cases with discrepancies between SS and NGS due to human errors in the mutation calling of the initial Sanger sequence are shown with 25 reads and two amino acids surrounding the affected amino acid are depicted. In addition, the allelic frequency (%) as well as coverage (x) is given. A, The peak representing the mutation from the electropherogram could not be assigned to its correct position for PIK3CA c.3140A>T p. H1047L because the primers are too close to the position of the mutation. B, A clear C to A substitution in the Sanger sequence is visible but was misread as a G to C substitution leading to the wrong amino acid substitution, that is, c.34_35GG>CC p. G12L instead of c.34_35delinsTT p. G12F. C, In one patient, the correct base substitution was noted but the wrong amino acid substitution reported. D and E, Representative case in which a T790M resistance mutation was found in EGFR exon 20, which was not screened for by the SS pipeline (D). The pathogenic co-occurring mutation in the same sample in EGFR exon 21: c.2573T>G p. L858R is shown in (E). NGS, next-generation sequencing; SS, Sanger sequencing. Journal of Thoracic Oncology 2015 10, 1049-1057DOI: (10.1097/JTO.0000000000000570) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Multiplexing leads to reduced turnaround time. A, The mean coverage for six runs on the MiSeq in a routine diagnostic is shown including the number of samples loaded (n), cluster density,49 and the Quality30 score. B, The number of days to complete full analysis by SS (left panel) and NGS (right panel). n is the number of patients analyzed. NGS, next-generation sequencing; SS, Sanger sequencing. Journal of Thoracic Oncology 2015 10, 1049-1057DOI: (10.1097/JTO.0000000000000570) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 NGS in routine diagnostics reveals expected mutation frequencies for EGFR, KRAS, and BRAF as well as an equal mutation rate for DDR2 in ADs and SCC. A and B, One thousand nine hundred seventy-one consecutive ADs and 686 SCCs were analyzed by NGS, which detected driver mutations in 87% of the samples with frequencies depicted here. DDR2 mutations were found with a frequency of 3% both in ADs and SCCs. C and D, Thirty-five percent of the ADs and 57% of the SCCs samples harbored TP53 mutations. All TP53 mutated cases and the frequency of co-occurring driver mutations are depicted. ADs, adenocarcinomas; SCC, squamous cell carcinoma. Journal of Thoracic Oncology 2015 10, 1049-1057DOI: (10.1097/JTO.0000000000000570) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions