Assessment of Capture and Amplicon-Based Approaches for the Development of a Targeted Next-Generation Sequencing Pipeline to Personalize Lymphoma Management

Publication date: March 2018 Source:The Journal of Molecular Diagnostics, Volume 20, Issue 2 Author(s): Stacy S. Hung, Barbara Meissner, Elizabeth A. Chavez, Susana Ben-Neriah, Daisuke Ennishi, Martin R. Jones, Hennady P. Shulha, Fong Chun Chan, Merrill Boyle, Robert Kridel, Randy D. Gascoyne, Andrew J. Mungall, Marco A. Marra, David W. Scott, Joseph M. Connors, Christian Steidl Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation ...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research