A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> We have enhanced the polymerase chain reaction (PCR) such that specific DNA sequences can be detected without opening the reaction tube. This enhancement requires the addition of ethidium bromide (EtBr) to a PCR. Since the fluorescence of EtBr increases in the presence of double-stranded (ds) DNA an increase in fluorescence in such a PCR indicates a positive amplification, which can be easily monitored externally. In fact, amplification can be continuously monitored in order to follow its progress. The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample throughput. <s> BIB001 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> BACKGROUND ::: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. ::: ::: ::: CONTENT ::: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. ::: ::: ::: SUMMARY ::: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results. <s> BIB002 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) 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. <s> BIB003 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> qPCR instruments are supplied with basic software packages that enable the measurement of fluorescent AQ1 AQ2 changes, calculations of quantification cycle (Cq) values, the generation of standard curves and subsequent relative target nucleic acid quantity determination. However, detailed assessments of the technical parameters underlying Cq values and their translation into biological meaningful results require validation of these basic calculations through further analyses such as qPCR efficiency correction, normalization to multiple reference genes, averaging and statistical tests. Some instruments incorporate some of these features, while others offer additional tools to complement the basic running software, in many cases providing those that are described below. In this chapter, there is a detailed description of some of these programs and recommended strategies for the design of robust qPCR assays. Some of the packages available for validation of the resulting Cq data and detailed statistical analysis are described. <s> BIB004 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> Scientific reproducibility now very often depends on the computational method being available to duplicate, so here it is argued that all source code should be freely available. Most scientific papers published today rely on computer programs for data collection and manipulation. Writing in the Perspective pages in this issue of Nature, Darrel Ince and colleagues argue that the policies of most journals and funding bodies towards the release of computer codes as part of the publication process are obsolete. They say that the full release of actual source code should be the norm for any scientific results dependent on computation, with an agreed list of exceptions applicable only to rare cases. Current policies range from a requirement for release of the relevant computer programs on request to Nature's less stringent stipulation of a 'natural language' description of computer algorithms. Scientific communication relies on evidence that cannot be entirely included in publications, but the rise of computational science has added a new layer of inaccessibility. Although it is now accepted that data should be made available on request, the current regulations regarding the availability of software are inconsistent. We argue that, with some exceptions, anything less than the release of source programs is intolerable for results that depend on computation. The vagaries of hardware, software and natural language will always ensure that exact reproducibility remains uncertain, but withholding code increases the chances that efforts to reproduce results will fail. <s> BIB005 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> The publication and open exchange of knowledge and material form the backbone of scientific progress and reproducibility and are obligatory for publicly funded research. Despite increasing reliance on computing in every domain of scientific endeavor, the computer source code critical to understanding and evaluating computer programs is commonly withheld, effectively rendering these programs “black boxes” in the research work flow. Exempting from basic publication and disclosure standards such a ubiquitous category of research tool carries substantial negative consequences. Eliminating this disparity will require concerted policy action by funding agencies and journal publishers, as well as changes in the way research institutions receiving public funds manage their intellectual property (IP). <s> BIB006 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references. <s> BIB007 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Introduction <s> This review (containing 101 refs.) covers recent achievements in the development of new approaches for enzymatically assisted detection of nucleic acids on microarrays. We discuss molecular techniques including the polymerase chain reaction, reverse transcription, allele specific primer extension and a range of isothermal techniques for the amplification and discrimination of nucleic acids. This also includes their implementation into microfluidic systems. These techniques all show great promise for use in the life sciences by allowing for high throughput, cost effective and highly sensitive and specific analysis of nucleic acids. Importantly, they can be potentially integrated into personalized and point-of-care medicine. <s> BIB008
Since its commercial introduction almost 2 decades ago, realtime quantitative polymerase-chain-reaction (qPCR) has come to play a prominent role in the life sciences. It provides the base for a plethora of applications in basic research, pathogen detection, and biomedical diagnostics. Furthermore, it is widely accepted as the gold standard for the analysis of gene expression. qPCR is a molecular biology technique, which allows amplification and simultaneous quantification of a targeted DNA molecule. The advancement compared to the original PCR method is the ability to measure the amplification of DNA as the reaction progresses in real time BIB001 . This allows quantifying initial amounts of template molecules by comparing the number of amplification cycles required for the response curves to reach a particular quantification threshold fluorescence signal level  . The more copies of a DNA template are present at the beginning of an experiment, the fewer PCR cycles are needed to make enough material for detection. In the past years, isothermal amplification strategies emerged as alternatives to PCR. Examples include the helicase-dependent isothermal DNA amplification (HDA), and the recombinase polymerase amplification (RPA). In contrast to traditional PCR, these methods do not require changing the reaction temperature and therefore use time-based measurements instead of cycle based measurements. However, isothermal amplification methods have less clinical use than conventional PCR BIB007 BIB008 . Since the commercialization of qPCR in 1996, the number of publications referring to qPCR has increased exponentially (see Fig. 1 ). To ensure quality of results and allow potential reproduction, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have been published BIB002 . They provide a basic set of quality criteria and detail 85 parameters ranging from experimental design over sample processing and assay validation to data analysis. After successfully generating a high-quality qPCR run, the data needs to be correctly analyzed to get biological meaningful results. To facilitate sharing and exchanging experimental data, the Realtime PCR Data Markup Language (RDML) has been developed BIB003 . The data standard is based on XML and contains details about the experimental setup, information about the samples and targets, and all measured data. As the data analysis step is an essential part of the qPCR workflow, it should be performed in a standardized and reproducible way BIB004 . As a consequence, dedicated software packages and data analysis suites were created targeting different aspects of the qPCR analysis workflow. Usually, commercial qPCR systems are equipped with software for data analysis and visualization. However, in most of the cases these closed source software modules are black boxes and do not provide full control over the entire process. Recently, Nature BIB005 and Science BIB006 reminded the scientific community about the importance of open computer programs. In this survey we reviewed 27 software packages and tools for the analysis of qPCR data. We targeted all available open-access tools capable of analyzing raw fluorescence or Cq (quantification cycle) values. First we provide an overview about the main application fields for qPCR. Next, we report an overview of the functionality, features and specific requirements of the individual tools. Finally, we briefly discuss the surveyed applications and provide a report on their provided functionalities.