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1.  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
2.  Measurable impact of RNA quality on gene expression results from quantitative PCR 
Nucleic Acids Research  2011;39(9):e63.
Compromised RNA quality is suggested to lead to unreliable results in gene expression studies. Therefore, assessment of RNA integrity and purity is deemed essential prior to including samples in the analytical pipeline. This may be of particular importance when diagnostic, prognostic or therapeutic conclusions depend on such analyses. In this study, the comparative value of six RNA quality parameters was determined using a large panel of 740 primary tumour samples for which real-time quantitative PCR gene expression results were available. The tested parameters comprise of microfluidic capillary electrophoresis based 18S/28S rRNA ratio and RNA Quality Index value, HPRT1 5′–3′ difference in quantification cycle (Cq) and HPRT1 3′ Cq value based on a 5′/3′ ratio mRNA integrity assay, the Cq value of expressed Alu repeat sequences and a normalization factor based on the mean expression level of four reference genes. Upon establishment of an innovative analytical framework to assess impact of RNA quality, we observed a measurable impact of RNA quality on the variation of the reference genes, on the significance of differential expression of prognostic marker genes between two cancer patient risk groups, and on risk classification performance using a multigene signature. This study forms the basis for further rational assessment of reverse transcription quantitative PCR based results in relation to RNA quality.
doi:10.1093/nar/gkr065
PMCID: PMC3089491  PMID: 21317187

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