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  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

Kohlmann, A., Klein, H. U., Weissmann, S., Bresolin, S., Chaplin, T., Cuppens, H., et al. (2011). 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: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K, 25(12), 1840-8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21681191 http://www.nature.com/leu/journal/v25/n12/pdf/leu2011155a.pdf.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-78F8-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-78F9-F
Genre: Journal Article

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 Creators:
Kohlmann, A., Author
Klein, H. U., Author
Weissmann, S., Author
Bresolin, S., Author
Chaplin, T., Author
Cuppens, H., Author
Haschke-Becher, E., Author
Garicochea, B., Author
Grossmann, V., Author
Hanczaruk, B., Author
Hebestreit, K., Author
Gabriel, C., Author
Iacobucci, I., Author
Jansen, J. H., Author
te Kronnie, G., Author
van de Locht, L., Author
Martinelli, G., Author
McGowan, K., Author
Schweiger, M. R.1, Author              
Timmermann, B.2, Author              
Vandenberghe, P., AuthorYoung, B. D., AuthorDugas, M., AuthorHaferlach, T., Author more..
Affiliations:
1Cancer Genomics (Michal-Ruth Schweiger), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479649              
2Sequencing, Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433559              

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Free keywords: Aged; Aged, 80 and over; Cohort Studies; DNA Mutational Analysis; DNA-Binding Proteins/*genetics; Female; *High-Throughput Nucleotide Sequencing; Humans; Leukemia, Myelomonocytic, Chronic/*genetics; Male; Middle Aged; Mutation/*genetics; Polymerase Chain Reaction; Prognosis; Proto-Oncogene Proteins/*genetics; Proto-Oncogene Proteins c-cbl/*genetics; ras Proteins/*genetics
 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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 Dates: 2011
 Publication Status: Published in print
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Title: Leukemia : Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
Source Genre: Journal
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Pages: - Volume / Issue: 25 (12) Sequence Number: - Start / End Page: 1840 - 8 Identifier: ISSN: 1476-5551 (Electronic) 0887-6924 (Linking)