Journal Article

Mate Pair Sequencing of Whole-Genome-Amplified DNA Following Laser Capture Microdissection of Prostate Cancer

Stephen J. Murphy, John C. Cheville, Shabnam Zarei, Sarah H. Johnson, Robert A. Sikkink, Farhad Kosari, Andrew L. Feldman, Bruce W. Eckloff, R. Jeffrey Karnes and George Vasmatzis

in DNA Research

Published on behalf of Kazusa DNA Research Institute

Volume 19, issue 5, pages 395-406
Published in print October 2012 | ISSN: 1340-2838
Published online September 2012 | e-ISSN: 1756-1663 | DOI:

Show Summary Details


High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.

Keywords: mate pair sequencing; whole-genome amplified, laser capture microdissection; prostrate cancer

Journal Article.  7141 words.  Illustrated.

Subjects: Genetics and Genomics

Users without a subscription are not able to see the full content. Please, subscribe or login to access all content.