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1.  CASP8 SNP D302H (rs1045485) Is Associated with Worse Survival in MYCN-Amplified Neuroblastoma Patients 
PLoS ONE  2014;9(12):e114696.
Background
Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis.
Patients and Methods
We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays.
Results and Conclusion
We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8 gene SNP D302H was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors.
doi:10.1371/journal.pone.0114696
PMCID: PMC4263607  PMID: 25502557
2.  Target enrichment using parallel nanoliter quantitative PCR amplification 
BMC Genomics  2014;15:184.
Background
Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high.
Results
We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform.
Conclusions
Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform’s promise as a targeted resequencing method for multi-gene routine sequencing diagnostics.
doi:10.1186/1471-2164-15-184
PMCID: PMC4234423  PMID: 24612714
Next generation sequencing; Target enrichment; Sequence capture; Quantitative PCR; NCI60; Mutation detection
3.  Cancer Gene Prioritization for Targeted Resequencing Using FitSNP Scores 
PLoS ONE  2012;7(3):e31333.
Background
Although the throughput of next generation sequencing is increasing and at the same time the cost is substantially reduced, for the majority of laboratories whole genome sequencing of large cohorts of cancer samples is still not feasible. In addition, the low number of genomes that are being sequenced is often problematic for the downstream interpretation of the significance of the variants. Targeted resequencing can partially circumvent this problem; by focusing on a limited number of candidate cancer genes to sequence, more samples can be included in the screening, hence resulting in substantial improvement of the statistical power. In this study, a successful strategy for prioritizing candidate genes for targeted resequencing of cancer genomes is presented.
Results
Four prioritization strategies were evaluated on six different cancer types: genes were ranked using these strategies, and the positive predictive value (PPV) or mutation rate within the top-ranked genes was compared to the baseline mutation rate in each tumor type. Successful strategies generate gene lists in which the top is enriched for known mutated genes, as evidenced by an increase in PPV. A clear example of such an improvement is seen in colon cancer, where the PPV is increased by 2.3 fold compared to the baseline level when 100 top fitSNP genes are sequenced.
Conclusions
A gene prioritization strategy based on the fitSNP scores appears to be most successful in identifying mutated cancer genes across different tumor entities, with variance of gene expression levels as a good second best.
doi:10.1371/journal.pone.0031333
PMCID: PMC3291573  PMID: 22396732
4.  Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics 
PLoS ONE  2011;6(9):e25531.
Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics.
doi:10.1371/journal.pone.0025531
PMCID: PMC3184136  PMID: 21980484

Results 1-4 (4)