A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Absolute quantification (digital PCR) <s> We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications. <s> BIB001 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Absolute quantification (digital PCR) <s> Polymerase chain reaction (PCR)-based assays can target either DNA (the genome) or RNA (the transcriptome). Targeting the genome generates robust data that are informative and, most importantly, generally applicable. This is because the information contained within the genome is context-independent; i.e., generally, every normal cell contains the same DNA sequence--the same mutations and polymorphisms. The transcriptome, on the other hand, is context-dependent; i.e., the mRNA complement and level varies with physiology, pathology, or development. This makes the information contained within the transcriptome intrinsically flexible and variable. If this variability is combined with the technical limitations inherent in any reverse-transcription (RT)-PCR assay, it can be difficult to achieve not just a technically accurate but a biologically relevant result. Template quality, operator variability, the RT step itself, and subjectivity in data analysis and reporting are just a few technical aspects that make real-time RT-PCR appear to be a fragile assay that makes accurate data interpretation difficult. There can be little doubt that in the future, transcriptome-based analysis will become a routine technique. However, for the time being it remains a research tool, and it is important to recognize the considerable pitfalls associated with transcriptome analysis, with the successful application of RTPCR depending on careful experimental design, application, and validation. <s> BIB002 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Absolute quantification (digital PCR) <s> Several of the next generation sequencers are limited in their sample preparation process by the need to make an absolute measurement of the number of template molecules in the library to be sequenced. As currently practiced, the practical effects of this requirement compromise sequencing performance, both by requiring large amounts of sample DNA and by requiring extra sequencing runs to be performed. We used digital PCR to quantitate sequencing libraries, and demonstrated its sensitivity and robustness by preparing and sequencing libraries from subnanogram amounts of bacterial and human DNA on the 454 and Solexa sequencing platforms. This assay allows absolute quantitation and eliminates uncertainties associated with the construction and application of standard curves. The digital PCR platform consumes subfemptogram amounts of the sequencing library and gives highly accurate results, allowing the optimal DNA concentration to be used in setting up sequencing runs without costly and time-consuming titration techniques. This approach also reduces the input sample requirement more than 1000-fold: from micrograms of DNA to less than a nanogram. <s> BIB003 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Absolute quantification (digital PCR) <s> As the less familiar cousin of quantitative PCR moves mainstream, researchers have more options to choose from. <s> BIB004 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Absolute quantification (digital PCR) <s> 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. <s> BIB005 </s> A survey of tools for the analysis of quantitative PCR (qPCR) data <s> Absolute quantification (digital PCR) <s> Droplet digital PCR shows greater precision and reproducibility but no consistent gain in sensitivity when compared to real-time PCR. <s> BIB006
Digital PCR (dPCR) is a novel method for precise quantification of nucleic acids [18] . Prior to PCR, the reaction mixture is divided into a very large number of separate tiny volumes, such that there is either zero or one target molecule present in any individual reaction. Currently available commercial systems can generate up to ten thousands (Bio-Rad, Life Technologies), fifty thousand (Fluidigm), and ten million (RainDance) droplets per experiment BIB004 . In effect, each reaction becomes binary and the discrete signals can be counted. After applying a Poisson correction to account for wells with more than one copy, the counts can be used to quantify the absolute number of DNA molecules in the sample BIB001 . dPCR can be used for absolute quantification without the need for standard curves BIB003 , and has been reported to have a higher accuracy BIB003 as well as robustness to amplification efficiency BIB002 compared to qPCR. Due to the rising interest in dPCR, separate digital MIQE guidelines have been published to assist researchers in designing, conducting, and analyzing their experiments BIB005 . Recently, a study systematically compared the performance of qPCR with that of dPCR. The authors could show by using a variety of targets in different sample backgrounds, that dPCR with droplets showed greater precision and more reproducible findings  than qPCR BIB006 .