Detection of Mutations in Myeloid Malignancies through Paired-Sample Analysis of Microdroplet-PCR Deep Sequencing Data

Publication date: September 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 5 Author(s): Donavan T. Cheng , Janice Cheng , Talia N. Mitchell , Aijazuddin Syed , Ahmet Zehir , Nana Yaa T. Mensah , Alifya Oultache , Khedoudja Nafa , Ross L. Levine , Maria E. Arcila , Michael F. Berger , Cyrus V. Hedvat Amplicon-based methods for targeted resequencing of cancer genes have gained traction in the clinic as a strategy for molecular diagnostic testing. An 847-amplicon panel was designed with the RainDance DeepSeq system, covering most exons of 28 genes relevant to acute myeloid leukemia and myeloproliferative neoplasms. We developed a paired-sample analysis pipeline for variant calling and sought to assess its sensitivity and specificity relative to a set of samples with previously identified mutations. Thirty samples with known mutations in JAK2, NPM1, DNMT3A, MPL, IDH1, IDH2, CEBPA, and FLT3, were profiled and sequenced to high depth. Variant calling using an unmatched Hapmap DNA control removed a substantial number of artifactual calls regardless of algorithm used or variant class. The removed calls were nonunique, had lower variant frequencies, and tended to recur in multiple unrelated samples. Analysis of sample replicates revealed that reproducible calls had distinctly higher variant allele depths and frequencies compared to nonreproducible calls. On the basis of these differences, filters on variant frequency were chosen to select for reprod...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research