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 Harry, Harry; Haschke-Becher, E; Garicochea, B; Grossmann, V; Hanczaruk, B; Hebestreit, K; Gabriel, C; Iacobucci, I; Jansen, JH; Te Kronnie, G; van de Locht, L; Martinelli, G; McGowan, K; Schweiger, MR; Timmermann, B; Vandenberghe, Peter; Young, BD; Dugas, M; Haferlach, T

doi:10.1038/leu.2011.155

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

Author:

Kohlmann, A

Klein, H-U ; Weissmann, S ; Bresolin, S ; Chaplin, T ; Cuppens Harry, Harry ; Haschke-Becher, E ; Garicochea, B ; Grossmann, V ; Hanczaruk, B ; Hebestreit, K ; Gabriel, C ; Iacobucci, I ; Jansen, JH ; Te Kronnie, G ; van de Locht, L ; Martinelli, G ; McGowan, K ; Schweiger, MR ; Timmermann, B ; Vandenberghe, Peter ; Young, BD ; Dugas, M ; Haferlach, T

Keywords:

Science & Technology, Life Sciences & Biomedicine, Oncology, Hematology, next-generation sequencing, amplicon, deep sequencing, 454, coverage, mutation analysis, CHRONIC MYELOMONOCYTIC LEUKEMIA, MYELOID-LEUKEMIA, HIGH-RESOLUTION, REVEALS, GENE, CLONES, RAS, Aged, Aged, 80 and over, Cohort Studies, DNA Mutational Analysis, DNA-Binding Proteins, Dioxygenases, Female, High-Throughput Nucleotide Sequencing, Humans, Leukemia, Myelomonocytic, Chronic, Male, Middle Aged, Mutation, Polymerase Chain Reaction, Prognosis, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Proto-Oncogene Proteins p21(ras), ras Proteins, 1103 Clinical Sciences, 1112 Oncology and Carcinogenesis, Immunology, 3201 Cardiovascular medicine and haematology, 3202 Clinical sciences, 3211 Oncology and carcinogenesis

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.Leukemia advance online publication, 17 June 2011; doi:10.1038/leu.2011.155.