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1.  RDML-Ninja and RDMLdb for standardized exchange of qPCR data 
BMC Bioinformatics  2015;16(1):197.
The universal qPCR data exchange file format RDML is today well accepted by the scientific community, part of the MIQE guidelines and implemented in many qPCR instruments. With the increased use of RDML new challenges emerge. The flexibility of the RDML format resulted in some implementations that did not meet the expectations of the consortium in the level of support or the use of elements.
In the current RDML version 1.2 the description of the elements was sharpened. The open source editor RDML-Ninja was released ( RDML-Ninja allows to visualize, edit and validate RDML files and thus clarifies the use of RDML elements. Furthermore RDML-Ninja serves as reference implementation for RDML and enables migration between RDML versions independent of the instrument software. The database RDMLdb will serve as an online repository for RDML files and facilitate the exchange of RDML data ( Authors can upload their RDML files and reference them in publications by the unique identifier provided by RDMLdb. The MIQE guidelines propose a rich set of information required to document each qPCR run. RDML provides the vehicle to store and maintain this information and current development aims at further integration of MIQE requirements into the RDML format.
The editor RDML-Ninja and the database RDMLdb enable scientists to evaluate and exchange qPCR data in the instrument-independent RDML format. We are confident that this infrastructure will build the foundation for standardized qPCR data exchange among scientists, research groups, and during publication.
PMCID: PMC4474546  PMID: 26087842
2.  Target enrichment using parallel nanoliter quantitative PCR amplification 
BMC Genomics  2014;15:184.
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.
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.
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.
PMCID: PMC4234423  PMID: 24612714
Next generation sequencing; Target enrichment; Sequence capture; Quantitative PCR; NCI60; Mutation detection
3.  Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform 
BMC Medical Genomics  2012;5:17.
Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.
In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.
We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.
PMCID: PMC3443074  PMID: 22607986
Deafness; Next generation sequencing; PCR based enrichment; Genetic diagnostics
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.
PMCID: PMC3184136  PMID: 21980484
5.  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.
PMCID: PMC3089491  PMID: 21317187
6.  Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline 
BMC Bioinformatics  2010;11:269.
Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions.
We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data.
We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.
PMCID: PMC2880033  PMID: 20487544
7.  External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data 
Nucleic Acids Research  2009;37(21):e138.
The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.
PMCID: PMC2790878  PMID: 19734345
8.  RDML: structured language and reporting guidelines for real-time quantitative PCR data 
Nucleic Acids Research  2009;37(7):2065-2069.
The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium ( to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results.
PMCID: PMC2673419  PMID: 19223324

Results 1-8 (8)