Targeted resequencing of candidate genes using selector probes

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R2 = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation

450DNA Methylation Array Analysis of Chronic Lymphocytic Leukaemia Reveals Global Methylation to be Relatively Stable Over Time and Remarkably Similar in Cells Derived From Resting and Proliferative Compartments

The advent of global investigations has begun to unravel the functional involvement of DNA methylation in leukemogenesis. Previously, we identified divergent chronic lymphocytic leukaemia (CLL) subgroups to have differential methylation patterns effecting key canonical pathway, proliferation and apoptotic genes. Despite these advances, in CLL cells, little to no knowledge exists regarding the extent to which DNA methylation changes with respect to time and exposure to different microenvironments. For the first time, using high-resolution 450K DNA methylation arrays, the DNA methylation profiles of paired diagnostic/follow-up samples from 9 IGHVmutated/ treated and 9 IGHV-unmutated/untreated patients, as well as 10 patient-matched peripheral blood (PB) and lymph node (LN) samples were investigated. On a larger scale to our previous 27K array study, we revealed 2239 CpG sites as differentially methylated between IGHV-mutated and unmutated CLL patients. Interestingly, the majority of sites were positioned outside CpG islands and promoters. Novel findings include differential DNA methylation of CpG sites within known differentially expressed CLL prognostic genes CLLU1 and LPL, where methylation was notably higher in IGHV-mutated cases. Additionally, genes occupying TGF-ß and NF-B/TNFR1 pathways and large numbers of polycomb targets were differentially methylated. Over time, few large recurrent DNA methylation changes were noted among the subgroups. Although a larger number of non-recurrent differentially methylated sites were identified in IGHV-unmutated relative to IGHV-mutated cases over time, these changes equated to a low