Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

Background

DNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, however, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon next generation sequencing (NGS). The techniques were evaluated for linearity, accuracy and precision.

Results

MethyLight qPCR displayed the best linearity across the range of tested samples. Observed methylation measured by MethyLight- and MSRE/MDRE-qPCR and -dPCR were not significantly different to expected values whilst bisulfite amplicon NGS analysis over-estimated methylation content. Bisulfite amplicon NGS showed good precision, whilst the lower precision of qPCR and dPCR analysis precluded discrimination of differences of < 25% in methylation status. A novel dPCR MethyLight assay is also described as a potential method for absolute quantification that simultaneously measures both sense and antisense DNA strands following bisulfite treatment.

Conclusions

Our findings comprise a comprehensive benchmark for the quantitative accuracy of key methods for methylation analysis and demonstrate their applicability to the quantification of circulating tumour DNA biomarkers by using sample concentrations that are representative of typical clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1174) contains supplementary material, which is available to authorized users.     

Computational and mathematical modeling of the effects of tailbeat frequency and flexural stiffness in swimming fish

In this paper we describe how we combine computational and mathematical models to form virtual fish to explore different hypotheses about the impact of centra. We show how we create simulation models using a combination of a mathematical model of a fish-like robot using caudal fin propulsion, a propulsion model, and an optimizer, to explore the impact of centra under various scenarios. The optimizer uses the mathematical model to construct valid configurations of the digital robot and uses the utility function and propulsion model to evaluate the performance of each configuration. The evaluations are used to explore the adaptive landscape and find high-performing configurations. Our results show that the high-performing configurations have both increased (flexural) stiffness of the tail and higher tailbeat frequencies.     

Digital microfluidic hydrogel microreactors for proteomics

Proteolytic digestion is an essential step in proteomic sample processing. While this step has traditionally been implemented in homogeneous (solution) format, there is a growing trend to use heterogeneous systems in which the enzyme is immobilized on hydrogels or other solid supports. Here, we introduce the use of immobilized enzymes in hydrogels for proteomic sample processing in digital microfluidic (DMF) systems. In this technique, preformed cylindrical agarose discs bearing immobilized trypsin or pepsin were integrated into DMF devices. A fluorogenic assay was used to optimize the covalent modification procedure for enzymatic digestion efficiency, with maximum efficiency observed at 31 μg trypsin in 2-mm diameter agarose gel discs. Gel discs prepared in this manner were used in an integrated method in which proteomic samples were sequentially reduced, alkylated, and digested, with all sample and reagent handling controlled by DMF droplet operation. Mass spectrometry analysis of the products revealed that digestion using the trypsin gel discs resulted in higher sequence coverage in model analytes relative to conventional homogenous processing. Proof-of-principle was demonstrated for a parallel digestion system in which a single sample was simultaneously digested on multiple gel discs bearing different enzymes. We propose that these methods represent a useful new tool for the growing trend toward miniaturization and automation in proteomic sample processing.     

Applying digital technology to promote active and healthy confinement lifestyle during pandemics in the elderly

Although recognized as effective measures to curb the spread of the COVID19 outbreak, social distancing and home confinement have generated a mental health burden with older adults who are considered to be more vulnerable to psychosocial strains. To date, the application of digital technologies in response to COVID-19 pandemic has been narrowed to public-health needs related to containment and mitigation. However, information and communications technology (ICT)-based initiatives directed toward prediction and prevention of psychosocial support are still limited. Given the power of digital health solutions to allow easy and accurate characterization and intervention for health and disease, as well as to flatten the COVID19 incidence curves in many countries, our ECLB-COVID19 consortium is highlighting the importance of providing innovative ICT-based solutions (ICT-COVID-Companion) to improve elderly physical and mental health, thereby preventing/dampening psychosocial strain during pandemics. Based on innovative approaches (e.g., emotional/social computing, open social platform, interactive coaching, gamification, fitness-tracker, internet of things) and smart digital solutions (smartwatch/smartphone), smart companions must provide safe personalised physical, mental and psychosocial health surveillance. Additionally, by delivering personalised multi-dimension crisis-oriented health recommendations, such innovative crisis-oriented solutions would help (i) facilitate a user’s adherence to active and healthy confinement lifestyle (AHCL), (ii) achieve a rapid psychosocial recovery in case of depression issues and (iii) enhance preparedness for eventual future pandemics.     

An evaluation of the analysis system of video transects used to sample subtidal epibiota

Two popular methods of benthic cover estimation (the point intercept technique with two sets of position points, and digital interactive color segmentation) were compared with an alternative method of digital cover estimation using Bezier curves as a tool for outlining the objects on an images and AutoCAD® software for the final evaluation of abundance. The comparison was done using still video images obtained from two 10-m transects on subtidal rocks off the Rimsky-Korsakov islands in the Sea of Japan (Russia). Ten rectangular sectors (1×0.4 m each) selected randomly within both video transects were analyzed. One-way ANOVA for repeated measurements was used to test the differences between methods. The point intercept technique differed significantly from both methods of digital estimation and had an essential positive bias. The Tukey multiple comparison test revealed the differences among digital estimation methods in the species, which have the complicated color with many contrast spots. The proposed approach using Bezier curves has an advantage over the interactive color segmentation if the objects under selection are lit at different levels or have a contrast coloration or are hidden by canopy organisms. Besides, estimation of cover in the field of AutoCAD® software is more precise and takes less time than that obtained using a scaled grid, available for automatically segmented species. The results showed that digital cover estimation using Bezier curves and AutoCAD® software is a convenient method for analyzing benthic samples at large spatial scales.     

Can bivalve veligers escape feeding currents of adult bivalves?

While the stock of introduced Pacific oysters (Crassostrea gigas) increased in the Oosterschelde estuary (SW Netherlands), so did the filtration pressure of all bivalve species together. In the same period, stocks of native bivalves declined slightly. The expansion of Pacific oysters in Dutch estuaries might be partially due to better abilities of their larvae to avoid or escape filtration, compared to larvae of native bivalves. In this context, escape and swimming abilities of Pacific oyster larvae and the larvae of the native blue mussel (Mytilus edulis) were compared.Swimming behaviour of C. gigas larvae and larvae of M. edulis was recorded in still water and in a suction current mimicking a bivalve feeding current, in a horizontal and in a vertical plane. Larval swimming behaviour in a suction flow field was reconstructed by subtracting local water movement vectors from the total movement of larvae, yielding movement paths due to larval swimming alone.Swimming speeds and the rate of displacement in vertical direction of C. gigas and M. edulis larvae were related to larval shell length, and to the pitch of up- or downward swimming.Larvae of both species did not show escape reactions in a suction flow field. With increasing shell length, larval swimming speeds of both species increased significantly. Swimming speeds of C. gigas larvae were significantly higher than swimming speeds of M. edulis larvae, resulting in a faster vertical displacement. The ability to migrate to more favourable water layers faster may offer C. gigas an advantage over native bivalves with slower swimming larvae.     

Inter- and intra-specific variation in the facial colours of <Emphasis Type="Italic">Perisesarma eumolpe</Emphasis> and <Emphasis Type="Italic">Perisesarma indiarum</Emphasis> (Crustacea: Brachyura: Sesarmidae)

The colour of the characteristic facial bands in the mangrove crabs Perisesarma eumolpe (De Man) and Perisesarma indiarum (Tweedie) were investigated in relation to species, sex and size. Quantitative red/blue/green (RBG) values extracted from close-up digital photographs of 27 P. indiarum and 48 P. eumolpe specimens revealed significant differences between species and sexes. Males of both species possessed facial bands characterized by a greater intensity of blue, whereas green was more pronounced in female facial bands. It is postulated that these colour differences play a role in intra-specific sexual recognition. There existed a significant positive relationship between size and the intensity of blue in the facial bands of females of both species, suggesting these traits function as an indicator of their maturity (and possibly body condition). In large males, facial band colours contrast strongly against the surrounding mudflat and may play an important role in conspecific signalling during territorial disputes or contests over access to females. Handling editor: K. Martens     

Digital imaging analysis of size and shape of wheat and pea upon heating under anoxic conditions as a function of the temperature

Three types of experiments were carried out. In the first series of experiments emmer wheat grains (Triticum dicoccum Schübl), var. AR, and peas (Pisum sativum L.), var. RE, were heated at temperatures ranging from 130–700 °C under controlled anoxic conditions for a maximum of 120 min. For each temperature a separate experiment was carried out in a pre-heated tube oven. Image acquisition was carried out on a flat bed scanner with a transparency adapter; for the analysis the image program IMAGE J 1.27 was used. Various size and shape factors are discussed and the definitions of the selected size and shape factors are given. The size and shape were measured and the mean calculated from thirty of the untreated specimens and the charred residues heated at each temperature. The results show a change of size and shape as a function of the temperature. The changes for wheat grains and peas are not identical. It is suggested that the presence of the pericarp enclosing the wheat grains causes this difference. In a second series of experiments it was shown that untreated grains of emmer wheat, bread wheat and macaroni wheat can be separated by measuring the shape, but size varies too much within each species, probably the result of different growing conditions, to be useful for separation purposes. As a result of heating at temperatures higher than 290 °C the shape of the three species becomes identical. This implies that the three species can no longer be separated after being heated, solely on the basis of size and shape. Finally, both emmer wheat grains enclosed by chaff and without chaff were heated under similar conditions. After removal of the chaff from the former, the size and shape of the grains were measured. The results show no significant difference in size and shape between both types.