Analysis of qPCR data by converting exponentially related Ct values into linearly related X0 values

A common method for calculating results from qPCR experiments is the comparative Ct method, also called the 2(-ΔΔCt) method. However, several assumptions are included in the 2(-ΔΔCt) method and standard statistical analyses are not directly applicable. Here, we describe a different method, the X(0) method, for result calculations and statistical analysis from qPCR experiments. The X(0) method differs from the 2(-ΔΔCt) method by introducing a conversion of the exponentially related Ct values into linearly related X(0) values, which represent the amount of starting material in a qPCR experiment. Results calculated by the X(0) method are illustrated for qPCR experiments with technical and biological replicates, including procedures to calculate standard deviations. Incorporation of primer efficiencies in calculations by the X(0) method is also described. Altogether, the X(0) method constitutes a very simple and accurate alternative to the 2(-ΔΔCt) method for result calculations from qPCR data.     

Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

ABSTRACT: BACKGROUND: Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. RESULTS: Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40 % depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. CONCLUSION: Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.     

Comparison of novel and existing tools for studying drug sensitivity against the hookworm Ancylostoma ceylanicum in vitro

The motility assay is the current gold standard for evaluating drug effects on hookworm larvae and adults, however, among other drawbacks the assay is time consuming, and prone to individual subjectivity. We evaluated six alternative in vitro assays, namely the feeding inhibition assay, the colourimetric AlamarBlue?, MTT formazan and acid phosphatase activity assays, as well as isothermal calorimetry and the xCELLigence System using Ancylostoma ceylanicum third-stage larvae, stimulated third-stage larvae and adults. The performances of the assays were compared to the motility assay using three standard drugs: albendazole, levamisole and ivermectin (100-1 μg/ml). None of the assays investigated offered an advantage over the motility assay, because they were all inapplicable to third-stage larvae, which were presumably metabolically and physically too inactive. Among all assays tested the xCELLigence System performed best on adult worms as the test was accurate, simple, required a minimal number of worms and offered the possibility for conducting a medium-throughput screening.     

Flexible automation of cell culture and tissue engineering tasks

Until now, the predominant use cases of industrial robots have been routine handling tasks in the automotive industry. In biotechnology and tissue engineering, in contrast, only very few tasks have been automated with robots. New developments in robot platform and robot sensor technology, however, make it possible to automate plants that largely depend on human interaction with the production process, e.g., for material and cell culture fluid handling, transportation, operation of equipment, and maintenance. In this paper we present a robot system that lends itself to automating routine tasks in biotechnology but also has the potential to automate other production facilities that are similar in process structure. After motivating the design goals, we describe the system and its operation, illustrate sample runs, and give an assessment of the advantages. We conclude this paper by giving an outlook on possible further developments.     

Quantification of Shiga toxin 2‐encoding bacteriophages,by real‐time PCR and correlation with phage infectivity

Aims: To evaluate a qPCR‐based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles. Methods and Results: An approach based on qPCR was applied to count Stx phages in five phage lysates of known titre. The number of viral particles from each phage lysate was determined by electron microscopy using latex spheres. The infectivity of the Stx phages was evaluated onto three bacterial host strains, by double agar layer assay and plaque blot hybridization. The number of phage particles detected by electron microscopy correlates with the number calculated by qPCR in all the phages assayed. The number of infectious phages was from 1 to 3 log₁₀ units below the numbers obtained by qPCR and electron microscopy. Conclusions: The approach allows accurate quantification of Stx phages with a high recovery. The number of infectious phages is always below the number of phage particles detected by qPCR. Significance and Impact of the Study: The qPCR method is a good approach to enumerate Stx phages. However, these results should be carefully considered when related to the number of infectious phages for each lysate that could be applied in real samples, because values of infectious particles are always below the number of Stx phages detected by qPCR.     

Identification of stable senescence‐associated reference genes

Cellular senescence is a state of permanent cell cycle arrest activated in response to damaging stimuli. Many hallmarks associated with senescent cells are measured by quantitative real‐time PCR (qPCR). As the selection of stable reference genes for interpretation of qPCR data is often overlooked, we performed a systematic review to understand normalization strategies entailed in experiments involving senescent cells. We found that, in violation of the Minimum Information for publication of qPCR Experiments (MIQE) guidelines, most reports used only one reference gene to normalize qPCR data, and that stability of the reference genes was either not tested or not reported. To identify new and more stable reference genes in senescent fibroblasts, we analyzed the Shapiro–Wilk normality test and the coefficient of variation per gene using in public RNAseq datasets. We then compared the new reference gene candidates with commonly used ones by using both RNAseq and qPCR data. Finally, we defined the best reference genes to be used universally or in a strain‐dependent manner. This study intends to raise awareness of the instability of classical reference genes in senescent cells and to serve as a first attempt to define guidelines for the selection of more reliable normalization methods.     

Use of real-time quantitative PCR to investigate root and gall colonisation by co-inoculated isolates of the nematophagous fungus Pochonia chlamydosporia

The fungus Pochonia chlamydosporia is a potential biological control agent for plant parasitic nematodes, but to date, there has been little investigation of interactions (competitive, antagonistic or synergistic) between different isolates that occur together on roots and nematode galls. Real-time quantitative PCR (qPCR) has greatly improved the study of many fungi in situ on plant and nematode hosts, but distinguishing closely related isolates remains difficult. In this study, primers to discriminate P. chlamydosporia var. chlamydosporia and P. chlamydosporia var. catenulata were used to measure the relative abundance of isolates of the two varieties when inoculated singly or together on tomato plants. Also, sequence-characterised amplified polymorphic regions were identified to distinguish two different isolates of P. chlamydosporia var. chlamydosporia . Individual 1-cm root segments and nematode galls were excised, DNA extracted and subjected to real-time qPCR with the discriminatory primers. The qPCR method proved sensitive and reproducible and demonstrated that roots and nematode galls were not uniformly colonised by the fungi. Results indicated that the P. chalmydosporia var. catenulata isolate was more abundant on roots and eggs than P. chlamydosporia var. chlamydosporia , but all the isolates infected a similar proportion of nematode eggs. There was an indication that the abundance of each fungal isolate was reduced in co-inoculation experiments compared with single inoculations, but the number of root segments and galls colonised was not statistically significantly different.     

PRaTo: a web-tool to select optimal primer pairs for qPCR

An essential pre-requisite to perform sound quantitative real-time polymerase chain reaction (qPCR) assays is to design outstanding primer pairs. This means they must have a good efficiency and be not prone to produce multiple amplicons or primer dimer products. To circumvent these issues, several softwares are available to help primer design. Although satisfactory computer-aided primer design tools are available for standard PCR, less efforts were done to provide specific methods for selection of optimal primer pairs for qPCR. We have developed PRaTo a web-based tool that enables checking and ranking of primers pairs for their attitude to perform optimally and reliably when used in qPCR experiments. PRaTo is available at http://prato.daapv.unipd.it.     

The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

In this study, we explored whether SYBR Green-based quantitative real-time PCR (qPCR) could be used to determine the copy number of a plasmid and whether the method was broadly applicable to chromosomally encoded genetic elements often occurring in multiple copies, such as rRNA genes and insertion sequences (IS). Three different template sources (whole cells, total DNA, and restriction-enzyme digested total DNA) derived from the bacterium Comamonas sp. strain JS46 were analyzed by qPCR using primer-pairs targeting plasmid pJS46 and three chromosomally encoded sequences (16S rDNA, ISCsp1, and IS1071). The difference between threshold cycle values, C(T), of amplicons targeting these elements and of an amplicon targeting the single-copy reference element mnbA (chromosomally encoded) was used to establish DeltaC(T). DeltaC(T) values were then used to derive copy number. For pJS46, qPCR analyses of whole cells and total DNA underestimated the copy number of pJS46 approximately 7-fold and approximately 2.5-fold, respectively, whereas copy number values derived from qPCR analyses of digested total DNA were comparable to those derived from Southern blot (SB) analyses. In contrast, for the chromosomally encoded elements, qPCR analyses of all three template sources gave copy number values that were virtually identical to or differed by approximately 2 from copy number values derived by SB analysis. These data indicate that qPCR can be used to estimate the copy number of various genetic elements but that the accuracy of qPCR-derived values is affected by the template source.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

Investigating the effect of increasing robot group sizes on the human psychophysiological state in the context of human–swarm interaction

We study the psychophysiological state of humans when exposed to robot groups of varying sizes. In our experiments, 24 participants are exposed sequentially to groups of robots made up of 1, 3 and 24 robots. We measure both objective physiological metrics (skin conductance level and heart rate), and subjective self-reported metrics (from a psychological questionnaire). These measures allow us to analyse the psychophysiological state (stress, anxiety, happiness) of our participants. Our results show that the number of robots to which a human is exposed has a significant impact on the psychophysiological state of the human and that higher numbers of robots provoke a stronger response.     

Socially intelligent robots: dimensions of human-robot interaction

Social intelligence in robots has a quite recent history in artificial intelligence and robotics. However, it has become increasingly apparent that social and interactive skills are necessary requirements in many application areas and contexts where robots need to interact and collaborate with other robots or humans. Research on human-robot interaction (HRI) poses many challenges regarding the nature of interactivity and 'social behaviour' in robot and humans. The first part of this paper addresses dimensions of HRI, discussing requirements on social skills for robots and introducing the conceptual space of HRI studies. In order to illustrate these concepts, two examples of HRI research are presented. First, research is surveyed which investigates the development of a cognitive robot companion. The aim of this work is to develop social rules for robot behaviour (a 'robotiquette') that is comfortable and acceptable to humans. Second, robots are discussed as possible educational or therapeutic toys for children with autism. The concept of interactive emergence in human-child interactions is highlighted. Different types of play among children are discussed in the light of their potential investigation in human-robot experiments. The paper concludes by examining different paradigms regarding 'social relationships' of robots and people interacting with them.     

Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration

Aims:  To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large-volume drinking-water samples concentrated by ultrafiltration (UF).
Methods and Results:  Drinking-water samples (100 l) were seeded with Bacillus anthracis , Cryptospordium parvum , Francisella tularensis , Salmonella Typhi , and Vibrio cholerae and concentrated by UF. Recoveries by traditional methods were variable between samples and between some replicates; recoveries were not determined by qPCR. Francisella tularensis and V. cholerae were detected in all 14 samples after UF, B. anthracis was detected in 13, and C. parvum was detected in 9 out of 14 samples. Numbers found by qPCR after UF were significantly or nearly related to those found by traditional methods for all organisms except for C. parvum . A qPCR assay for S. Typhi was not available.
Conclusions:  qPCR can be used to rapidly detect biological agents after UF as well as traditional methods, but additional work is needed to improve qPCR assays for several biological agents, determine recoveries by qPCR, and expand the study to other areas.
Significance and Impact of the Study:  To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples.     

Sample degradation leads to false-positive copy number variation calls in multiplex real-time polymerase chain reaction assays

The recent implication of genomic copy number variations (CNVs) in multiple human genetic disorders has led to increased interest in CNV discovery technologies. There is a growing consensus that, in addition to the method used for detection, at least one additional technology should be employed for validation. Real-time quantitative polymerase chain reaction (qPCR) analysis, incorporating a normal (2N) copy number standard, is commonly used as a means of validating CNVs. Whereas it has previously been reported that formalin-fixed paraffin-embedded (FFPE) DNA samples can yield spurious CNV calls in real-time qPCR assays, here we report that sample degradation under standard laboratory storage conditions generates a significant increase in false-positive CNV results. Results suggest the possibility of biased degradation among genomic regions and emphasize the need to assess sample integrity immediately prior to real-time qPCR experiments.     

ARGoS: a modular, parallel, multi-engine simulator for multi-robot systems

We present a novel multi-robot simulator named ARGoS. ARGoS is designed to simulate complex experiments involving large swarms of robots of different types. ARGoS is the first multi-robot simulator that is at the same time both efficient (fast performance with many robots) and flexible (highly customizable for specific experiments). Novel design choices in ARGoS have enabled this breakthrough. First, in ARGoS, it is possible to partition the simulated space into multiple sub-spaces, managed by different physics engines running in parallel. Second, ARGoS?? architecture is multi-threaded, thus designed to optimize the usage of modern multi-core CPUs. Finally, the architecture of ARGoS is highly modular, enabling easy addition of custom features and appropriate allocation of computational resources. We assess the efficiency of ARGoS and showcase its flexibility with targeted experiments. Experimental results demonstrate that simulation run-time increases linearly with the number of robots. A 2D-dynamics simulation of 10,000 e-puck robots can be performed in 60?% of the time taken by the corresponding real-world experiment. We show how ARGoS can be extended to suit the needs of an experiment in which custom functionality is necessary to achieve sufficient simulation accuracy. ARGoS is open source software licensed under GPL3 and is downloadable free of charge.     

Hybridization methods for DNA sequencing.

I have conducted a general analysis of the practicability of using oligonucleotide hybridization to sequence DNA. Any DNA sequence may be sequenced by hybridization with a complete panel of oligonucleotides. However, sequencing DNA segments over 2 kb long requires an unrealistic number of hybridization reactions. The optimal protocol is to hybridize 7-mer or 8-mer mixed oligonucleotide probes to immobilized DNA fragments 80 bp long: should this prove impractical, hybridization of labeled 270-bp fragments to immobilized mixed 10-mers is a potential alternative. Both protocols require no more experiments to sequence large regions of DNA than conventional m13-based sequencing and are much easier to automate, thus reducing the requirements for skilled personnel. In the ideal case, hybridization sequencing reduces the number of experiments required to sequence megabase DNA by 90%.     

Self-organized flocking in mobile robot swarms

In this paper, we study self-organized flocking in a swarm of mobile robots. We present Kobot, a mobile robot platform developed specifically for swarm robotic studies. We describe its infrared-based short range sensing system, capable of measuring the distance from obstacles and detecting kin robots, and a novel sensing system called the virtual heading system (VHS) which uses a digital compass and a wireless communication module for sensing the relative headings of neighboring robots. We propose a behavior based on heading alignment and proximal control that is capable of generating self-organized flocking in a swarm of Kobots. By self-organized flocking we mean that a swarm of mobile robots, initially connected via proximal sensing, is able to wander in an environment by moving as a coherent group in open space and to avoid obstacles as if it were a “super-organism”. We propose a number of metrics to evaluate the quality of flocking. We use a default set of behavioral parameter values that can generate acceptable flocking in robots, and analyze the sensitivity of the flocking behavior against changes in each of the parameters using the metrics that were proposed. We show that the proposed behavior can generate flocking in a small group of physical robots in a closed arena as well as in a swarm of 1000 simulated robots in open space. We vary the three main characteristics of the VHS, namely: (1) the amount and nature of noise in the measurement of heading, (2) the number of VHS neighbors, and (3) the range of wireless communication. Our experiments show that the range of communication is the main factor that determines the maximum number of robots that can flock together and that the behavior is highly robust against the other two VHS characteristics. We conclude by discussing this result in the light of related theoretical studies in statistical physics.     

Task partitioning in swarms of robots: an adaptive method for strategy selection

Task partitioning is the decomposition of a task into two or more sub-tasks that can be tackled separately. Task partitioning can be observed in many species of social insects, as it is often an advantageous way of organizing the work of a group of individuals. Potential advantages of task partitioning are, among others: reduction of interference between workers, exploitation of individuals?? skills and specializations, energy efficiency, and higher parallelism. Even though swarms of robots can benefit from task partitioning in the same way as social insects do, only few works in swarm robotics are dedicated to this subject. In this paper, we study the case in which a swarm of robots has to tackle a task that can be partitioned into a sequence of two sub-tasks. We propose a method that allows the individual robots in the swarm to decide whether to partition the given task or not. The method is self-organized, relies on the experience of each individual, and does not require explicit communication between robots. We evaluate the method in simulation experiments, using foraging as testbed. We study cases in which task partitioning is preferable and cases in which it is not. We show that the proposed method leads to good performance of the swarm in both cases, by employing task partitioning only when it is advantageous. We also show that the swarm is able to react to changes in the environmental conditions by adapting the behavior on-line. Scalability experiments show that the proposed method performs well across all the tested group sizes.