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Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing
Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of rele...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519100/ https://www.ncbi.nlm.nih.gov/pubmed/26222071 http://dx.doi.org/10.1371/journal.pone.0133930 |
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author | Bartels, Stephan Schipper, Elisa Kreipe, Hans Heinrich Lehmann, Ulrich |
author_facet | Bartels, Stephan Schipper, Elisa Kreipe, Hans Heinrich Lehmann, Ulrich |
author_sort | Bartels, Stephan |
collection | PubMed |
description | Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies) was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17) as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10). Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41) were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43) could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., TET2, ASXL1, DNMT3A), demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons strongly argue for replacing Sanger sequencing by this semiconductor-based targeted resequencing approach. |
format | Online Article Text |
id | pubmed-4519100 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-45191002015-07-31 Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing Bartels, Stephan Schipper, Elisa Kreipe, Hans Heinrich Lehmann, Ulrich PLoS One Research Article Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies) was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17) as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10). Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41) were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43) could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., TET2, ASXL1, DNMT3A), demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons strongly argue for replacing Sanger sequencing by this semiconductor-based targeted resequencing approach. Public Library of Science 2015-07-29 /pmc/articles/PMC4519100/ /pubmed/26222071 http://dx.doi.org/10.1371/journal.pone.0133930 Text en © 2015 Bartels et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Bartels, Stephan Schipper, Elisa Kreipe, Hans Heinrich Lehmann, Ulrich Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing |
title | Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing |
title_full | Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing |
title_fullStr | Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing |
title_full_unstemmed | Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing |
title_short | Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing |
title_sort | comprehensive molecular profiling of archival bone marrow trephines using a commercially available leukemia panel and semiconductor-based targeted resequencing |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519100/ https://www.ncbi.nlm.nih.gov/pubmed/26222071 http://dx.doi.org/10.1371/journal.pone.0133930 |
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