[1879] Detection of Prognostically Significant AML Translocations and Single Base Mutations by Next Generation Sequencing.

Eric J Duncavage, Haley J Abel, Sarah South, John D Pfeifer. University of Utah, Salt Lake City; Washington University, St Louis, MO

Background: Acute Myeloid Leukemia (AML) is classified by the presence of recurrent cytogenetic abnormalities. Current methods for detection of these abnormalities offer relatively low sensitivity, and are often unable to define cryptic or uncommon translocation partners. Correlation with molecular studies for mutations in specific genes is also recommended, adding to testing complexity. We developed a targeted next generation sequencing (NGS)-based approach for clinical AML samples to identify both single gene mutations and translocations, including those with unique partners.
Design: Biotinylated RNA capture probes were designed to tile across introns and exons of genes implicated in AML risk assessment and comprised a target area of 1Mb. Patient genomic DNA, extracted from bone marrow of AML patients with known recurrent cytogenetic abnormalities, was captured and sequenced on an Illumina GAII using 60bp paired-end reads. Using DNA barcodes we sequenced 4 samples in multiplex on a single GAII lane. The resulting sequence data were then aligned to the targeted gene regions and translocations/large scale structural variations identified by the SLOPE software package.
Results: We previously sequenced one AML case with a known t(9:11) translocation in a single GAII lane, resulting in 1,100x coverage and successful identification of the translocation. In contrast, when the assay was multiplexed with 4 samples the average coverage became highly variable, ranging from 900x to 20x. We identified putative translocations in only the two cases with the greatest coverage, while mutations in single genes such as FLT3, NPM1, and CEBPA were identifiable in all cases.
Conclusions: Single and multiplex targeted NGS can be used to identify translocations and prognostically significant single gene mutations in AML. While translocations are easily detected in singly sequenced specimens, the current results demonstrate that the cost savings of multiplexed sequencing are offset by decreased and variable coverage, resulting in an inability to identify translocations in some cases. The data indicate, however, that longer read lengths will overcome these problems.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer

Monday, February 28, 2011 1:00 PM

Poster Session II # 240, Monday Afternoon


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