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

Abstract

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.

Integrated analysis of the genomic instability of PTEN in clinically insignificant and significant prostate cancer

“…[27][28][29][30] In Gleason score 7, the Gleason pattern 3 and Gleason pattern 4 were collected and analyzed separately. 28 Libraries were prepared using the Illumina Mate Pair (MP) Kit following the manufacturer's instructions and sequenced as two libraries per lane on the Illumina HiSeq platform.…”

“…28 Libraries were prepared using the Illumina Mate Pair (MP) Kit following the manufacturer's instructions and sequenced as two libraries per lane on the Illumina HiSeq platform. [27][28][29][30][31] Data were processed using previously described binary indexing mapping algorithms developed in our group. 32 The read-to-reference-genome-mapping algorithm was modified to map both mate pair sequencing reads across the whole genome.…”

Chromosomal rearrangements and copy number abnormalities of TP63 correlate with p63 protein expression in lung adenocarcinoma

“…Whole-genome amplification (WGA) was performed directly on laser captured microdissected cells using a single-step procedure. 22 Briefly, laser captured microdissected cells were incubated for 10 min in 0.5X Repli-g D2 buffer (6.5 ml) (Qiagen, CA, USA) and then in Repli-g stop solution (3.5 ml). Repli-g mini kit Master Mix (40 ml) was then added and incubated at 30 1C for 16 h. Four individual 50 ml WGA reactions were pooled for each sample.…”

“…DNA was quantified by Quant-iT PicoGreen analysis (Invitrogen, P7581). Mate-Pair libraries were assembled from WGA DNA according to a previously published protocol 22 using the Illumina mate-pair kit. Briefly, WGA DNA (10 mg) was fragmented to B3 kb and DNA intramolecularcircles assembled by ligation following biotin-end labeling.…”

2D and 3D Chromosome Painting in Malaria Mosquitoes

“…When coupled with whole genome amplification (WGA), microdissection results in powerful downstream applications including FISH 11,12 and next-generation genome sequencing [25][26][27] . Previously, the technique required the use of specialized microdissection needles that had to be manually controlled by an experienced user 28 .…”