High-throughput PCR assay design for targeted resequencing using primerXL.

BACKGROUND: Although the sequencing landscape is rapidly evolving and sequencing costs are continuously decreasing, whole genome sequencing is still too expensive for use on a routine basis. Targeted resequencing of only the regions of interest decreases both costs and the complexity of the downstream data-analysis. Various target enrichment strategies are available, but none of them obtain the degree of coverage uniformity, flexibility and specificity of PCR-based enrichment. On the other hand, the biggest limitation of target enrichment by PCR is the need to design large numbers of partially overlapping assays to cover the target. RESULTS: To overcome the aforementioned hurdles, we have developed primerXL, a state-of-the-art PCR primer design pipeline for targeted resequencing. It uses an optimized design criteria relaxation cascade and a thorough downstream in silico evaluation process to generate high quality singleplex PCR assays, reducing the need for amplicon normalization, and outperforming other target enrichment strategies and similar primer design tools when considering assay quality, coverage uniformity and target coverage. Results of four different sequencing projects with 2348 amplicons in total covering 470 kb are presented. PrimerXL can be accessed at www.primerxl.org . CONCLUSION: PrimerXL is an state-of-the-art, easy to use primer design webtool capable of generating high-quality targeted resequencing assays. The workflow is fully customizable to suit every researchers' needs, while an innovative relaxation cascade ensures maximal target coverage.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

RDML-Ninja and RDMLdb for standardized exchange of qPCR data.

BACKGROUND: 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. RESULTS: In the current RDML version 1.2 the description of the elements was sharpened. The open source editor RDML-Ninja was released (http://sourceforge.net/projects/qpcr-ninja/). 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 (http://www.rdmldb.org). 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. CONCLUSIONS: 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.

Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice.

The release of benchtop next-generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost-efficient workflows is high. We used singleplex-PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing-based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost-efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189-accreditation was obtained for the workflow. A major asset of the singleplex PCR-based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases.

Target enrichment using parallel nanoliter quantitative PCR amplification.

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.

Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications.

RNA transcripts such as mRNA or microRNA are frequently used as biomarkers to determine disease state or response to therapy. Reverse transcription (RT) in combination with quantitative PCR (qPCR) has become the method of choice to quantify small amounts of such RNA molecules. In parallel with the democratization of RT-qPCR and its increasing use in biomedical research or biomarker discovery, we witnessed a growth in the number of gene expression data analysis methods. Most of these methods are based on the principle that the position of the amplification curve with respect to the cycle-axis is a measure for the initial target quantity: the later the curve, the lower the target quantity. However, most methods differ in the mathematical algorithms used to determine this position, as well as in the way the efficiency of the PCR reaction (the fold increase of product per cycle) is determined and applied in the calculations. Moreover, there is dispute about whether the PCR efficiency is constant or continuously decreasing. Together this has lead to the development of different methods to analyze amplification curves. In published comparisons of these methods, available algorithms were typically applied in a restricted or outdated way, which does not do them justice. Therefore, we aimed at development of a framework for robust and unbiased assessment of curve analysis performance whereby various publicly available curve analysis methods were thoroughly compared using a previously published large clinical data set (Vermeulen et al., 2009) [11]. The original developers of these methods applied their algorithms and are co-author on this study. We assessed the curve analysis methods' impact on transcriptional biomarker identification in terms of expression level, statistical significance, and patient-classification accuracy. The concentration series per gene, together with data sets from unpublished technical performance experiments, were analyzed in order to assess the algorithms' precision, bias, and resolution. While large differences exist between methods when considering the technical performance experiments, most methods perform relatively well on the biomarker data. The data and the analysis results per method are made available to serve as benchmark for further development and evaluation of qPCR curve analysis methods (http://qPCRDataMethods.hfrc.nl).

A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2.

We report heterozygous mutations in the genes encoding either type I or type II transforming growth factor beta receptor in ten families with a newly described human phenotype that includes widespread perturbations in cardiovascular, craniofacial, neurocognitive and skeletal development. Despite evidence that receptors derived from selected mutated alleles cannot support TGFbeta signal propagation, cells derived from individuals heterozygous with respect to these mutations did not show altered kinetics of the acute phase response to administered ligand. Furthermore, tissues derived from affected individuals showed increased expression of both collagen and connective tissue growth factor, as well as nuclear enrichment of phosphorylated Smad2, indicative of increased TGFbeta signaling. These data definitively implicate perturbation of TGFbeta signaling in many common human phenotypes, including craniosynostosis, cleft palate, arterial aneurysms, congenital heart disease and mental retardation, and suggest that comprehensive mechanistic insight will require consideration of both primary and compensatory events.

Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin.

Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1-4 (refs. 4-9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10-20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.

Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays.

BACKGROUND: Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility. METHODS: We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield. RESULTS: The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3' end of the primer. In addition, having ≥4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased. CONCLUSIONS: Single mismatches located >5 bp from the 3' end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for ≥4 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.

The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

Measurable impact of RNA quality on gene expression results from quantitative PCR.

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.

Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis.

Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Homozygous inactivating mutations in the NKX3-2 gene result in spondylo-megaepiphyseal-metaphyseal dysplasia.

Spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD) is a rare skeletal dysplasia with only a few cases reported in the literature. Affected individuals have a disproportionate short stature with a short and stiff neck and trunk. The limbs appear relatively long and may show flexion contractures of the distal joints. The most remarkable radiographic features are the delayed and impaired ossification of the vertebral bodies as well as the presence of large epiphyseal ossification centers and wide growth plates in the long tubular bones. Numerous pseudoepiphyses of the short tubular bones in hands and feet are another remarkable feature of the disorder. Genome wide homozygosity mapping followed by a candidate gene approach resulted in the elucidation of the genetic cause in three new consanguineous families with SMMD. Each proband was homozygous for a different inactivating mutation in NKX3-2, a homeobox-containing gene located on chromosome 4p15.33. Striking similarities were found when comparing the vertebral ossification defects in SMMD patients with those observed in the Nkx3-2 null mice. Distinguishing features were the asplenia found in the mutant mice and the radiographic abnormalities in the limbs only observed in SMMD patients. The absence of the latter anomalies in the murine model may be due to the perinatal death of the affected animals. This study illustrates that NKX3-2 plays an important role in endochondral ossification of both the axial and appendicular skeleton in humans. In addition, it defines SMMD as yet another skeletal dysplasia with autosomal-recessive inheritance and a distinct phenotype.

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes. METHODS: We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause. RESULTS: Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations. CONCLUSION: We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.

Evaluation of efficiency and sensitivity of 1D and 2D sample pooling strategies for SARS-CoV-2 RT-qPCR screening purposes.

To increase the throughput, lower the cost, and save scarce test reagents, laboratories can pool patient samples before SARS-CoV-2 RT-qPCR testing. While different sample pooling methods have been proposed and effectively implemented in some laboratories, no systematic and large-scale evaluations exist using real-life quantitative data gathered throughout the different epidemiological stages. Here, we use anonymous data from 9673 positive cases to model, simulate and compare 1D and 2D pooling strategies. We show that the optimal choice of pooling method and pool size is an intricate decision with a testing population-dependent efficiency-sensitivity trade-off and present an online tool to provide the reader with custom real-time 1D pooling strategy recommendations.

Consensus guidelines for the validation of qRT-PCR assays in clinical research by the CardioRNA consortium.

Despite promising findings, quantitative PCR (qPCR)-based tests for RNA quantification have experienced serious limitations in their clinical application. The noticeable lack of technical standardization remains a huge obstacle in the translation of qPCR-based tests. The incorporation of qPCR-based tests into the clinic will benefit from guidelines for clinical research assay validation. This will ultimately impact the clinical management of the patient, including diagnosis, prognosis, prediction, monitoring of the therapeutic response, and evaluation of toxicity. However, clear assay validation protocols for biomarker investigation in clinical trials using molecular assays are currently lacking. Here, we will focus on the necessary steps, including sample acquisition, processing and storage, RNA purification, target selection, assay design, and experimental design, that need to be taken toward the appropriate validation of qRT-PCR assays in clinical research. These recommendations can fill the gap between research use only (RUO) and in vitro diagnostics (IVD). Our contribution provides a tool for basic and clinical research for the development of validated assays in the intermediate steps of biomarker research. These guidelines are based on the current understanding and consensus within the EU-CardioRNA COST Action consortium (www.cardiorna.eu). Their applicability encompasses all clinical areas.

Reliable and Scalable SARS-CoV-2 qPCR Testing at a High Sample Throughput: Lessons Learned from the Belgian Initiative.

We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country's testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.

Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2: opportunities, challenges, and limitations.

This study describes how the new massive parallel sequencing technology can be implemented in a diagnostic setting for the breast cancer susceptibility genes (BRCA1 and BRCA2). The throughput was maximized by increasing uniformity in coverage, obtained by a multiplex approach, which outperformed pooling of singleplex PCRs. We evaluated the sensitivity by analysis of 133 distinct sequence variants; three (2%) deletions or duplications in homopolymers of greater than or equal to seven nucleotides remained undetected, illustrating a limitation of pyrosequencing. Furthermore, other limitations like nonrandom sequencing errors, pseudogene amplification, and failure to detect multiexon deletions are thoroughly described. Our workflow illustrates the potential of massive parallel sequencing of large genes in a diagnostic setting, which is of great importance to meet the increasing expectations of genetic testing. Implementation of this approach will hopefully lead to a strong reduction in turnaround times. As a consequence a wider spectrum of at risk women will be able to benefit from therapeutic interventions and prophylactic interventions.

Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes.

The Marfan (MFS) and Loeys-Dietz (LDS) syndromes are caused by mutations in the fibrillin-1 (FBN1) and Transforming Growth Factor Beta Receptor 1 and 2 (TGFBR1 and TGFBR2) genes, respectively. With the current conventional mutation screening technologies, analysis of this set of genes is time consuming and expensive. We have tailored a cost-effective and reliable mutation discovery strategy using multiplex PCR followed by Next Generation Sequencing (NGS). In a first stage, genomic DNA from five MFS or LDS patient samples with previously identified mutations and/or polymorphisms in FBN1 and TGFBR1 and 2 were analyzed and revealed all expected variants. In a second stage, we validated the technique on 87 samples from MFS patients fulfilling the Ghent criteria. This resulted in the identification of 75 FBN1 mutations, of which 67 were unique. Subsequent Multiplex Ligation-dependent Probe Amplification (MLPA) analysis of the remaining negative samples identified four large deletions/insertions. Finally, Sanger sequencing identified a missense mutation in FBN1 exon 1 that was not included in the NGS workflow. In total, there was an overall mutation identification rate of 92%, which is in agreement with data published previously. We conclude that multiplex PCR of all coding exons of FBN1 and TGFBR1/2 followed by NGS analysis and MLPA is a robust strategy for time- and cost-effective identification of mutations.