Abstract
Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon next-generation deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1–2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.
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Acknowledgements
Study supplies and training courses were provided by Roche Diagnostics GmbH Penzberg, Germany. We would like to thank Verena Lutz, Bruno Frey and Wulf Fischer-Knuppertz for their support throughout the conduct of the study. Research in this work was further supported by the European LeukemiaNet (Work Package 13).
Author Contribution
AK and TH designed the study. SW, SB, TC, HC, EH-B, BG, VG, BH, CG, II, JHJ, GtK, LvdL, GM, KM, MRS, BT, PV and BDY performed research and generated data. H-UK, SW, KH, MD and AK analyzed and interpreted the data. AK, H-UK and SW wrote the paper.
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TH is a part-owner of the MLL Munich Leukemia Laboratory GmbH. AK, SW and VG are employed by MLL Munich Leukemia Laboratory GmbH. AK has received honoraria from Roche Diagnostics GmbH Mannheim, Germany. Other authors declare no conflict of interest.
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Kohlmann, A., Klein, HU., Weissmann, S. et al. The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories. Leukemia 25, 1840–1848 (2011). https://doi.org/10.1038/leu.2011.155
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DOI: https://doi.org/10.1038/leu.2011.155
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