Impact of variance components on reliability of absolute quantification using digital PCR

Background

Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation.

Results

We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably.

Conclusions

Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-283) contains supplementary material, which is available to authorized users.     

Mapping mountain vegetation using species distribution modeling,image‐based texture analysis,and object‐based classification

Objective: The objective of this study was to map vegetation composition across a 24 000 ha watershed. Location: The study was conducted on the western slope of the Sierra Nevada mountain range of California, USA. Methods: Automated image segmentation was used to delineate image objects representing vegetation patches of similar physiognomy and structure. Image objects were classified using a decision tree and data sources extracted from individual species distribution models, Landsat spectral data, and life form cover estimates derived from aerial image‐based texture variables. Results: A total of 12 plant communities were mapped with an overall accuracy of 75% and a χ‐value of 0.69. Species distribution model inputs improved map accuracy by approximately 15% over maps derived solely from image data. Automated mapping of existing vegetation distributions, based solely on predictive distribution model results, proved to be more accurate than mapping based on Landsat data, and equivalent in accuracy to mapping based on all image data sources. Conclusions: Results highlight the importance of terrain, edaphic, and bioclimatic variables when mapping vegetation communities in complex terrain. Mapping errors stemmed from the lack of spectral discernability between vegetation classes, and the inability to account for the confounding effects of land use history and disturbance within a static distribution modeling framework.     

Brazilian Páramos IV. Phytogeography of the campos de altitude

Aim This contribution treats the phytogeography of the contemporary campos de altitude flora, with a focus on patterns at the level of genus. Comparative analysis using data from 17 other sites in Latin America is used to describe phytogeographical patterns at the continental scale. Results are combined with those of previous publications to shed light on the biogeographical origins of contemporary floristic patterns in the high mountains of south‐east Brazil. Location The campos de altitude are a series of cool‐humid, mountaintop grass‐ and shrublands found above elevations of 1800–2000 m in south‐east Brazil, within the biome of the Atlantic Forest. Methods Vascular floras are compiled for the three best‐known campos de altitude sites, and for 17 other highland and lowland locations in Latin America. Floras are binned into phytogeographical groups based on centres of diversity/origin. Floristic and geographical distances are calculated for all location‐pairs; Mantel tests are used to test for relationships between patterns in geographical distance, and floristic and climatic similarity. Multivariate statistics are carried out on the similarity matrices for all genera, and for each phytogeographical group. Predominant life‐forms, pollination and dispersal syndromes are determined for each genus in the campos de altitude flora, and proportional comparisons are made between phytogeographical groups. Supporting evidence from previously published literature is used to interpret analytical results. Results Two‐thirds of the genera in the campos de altitude are of tropical ancestry; the remainder are of temperate‐zone or cosmopolitan ancestry. Most campos de altitude genera are phanerophytes and hemicryptophytes, insect pollinated, and wind or gravity dispersed, but there are significant differences in the distribution of these traits among phytogeographical groups. The campos de altitude show stronger floristic similarities with other Brazilian mountain sites and distant Andean sites than with nearby low‐ and middle‐elevation sites; these similarities are best explained by climatic similarities. Floristic similarities among sites for temperate genera are better explained by ‘sinuous’ distance (e.g. measured along the spines of mountain ranges) than by direct distance; similarities in tropical genera are more related to direct distance. Different phytogeographical groups appear to be responding to different climatic signals. Main conclusions Many taxa currently living at the summits of the south‐east Brazilian Highlands trace their ancestry to temperate latitudes. Patterns of endemism and diversity in the south‐east Brazilian mountains point to climatically driven allopatry as a principal mechanism for speciation. The tropical component of the campos de altitude flora is primarily derived from drier, highland environments of the Brazilian interior; the temperate component rises in importance with elevation, but never reaches the levels seen in the tropical Andes. Most temperate taxa in the campos de altitude appear to have arrived via migration through favourable habitat rather than by recent, long‐distance dispersal. At least 11% of the plant species in the campos de altitude study sites are directly shared with the Andes. Palynofloras show that the campos de altitude have significantly contracted over the past 10,000 years, as regional temperatures have warmed and become more humid.     

Does Auxin Binding Protein 1 Control Both Cell Division and Cell Expansion?

The Auxin-Binding Protein 1 (ABP1) was identified over 30 years ago thanks to it''s high affinity for active auxins. ABP1 plays an essential role in plant life yet to this day, its function remains ‘enigmatic.’ A recent study by our laboratory shows that ABP1 is critical for regulation of the cell cycle, acting both in G₁ and at the G₂/M transition. We showed that ABP1 is likely to mediate the permissive auxin signal for entry into the cell cycle. These data were obtained by studying a conditional functional knock-out of ABP1 generated by cellular immunization in the model tobacco cell line, Bright Yellow 2.Key Words: auxin responses, auxin-binding protein 1, immunomodulation, cellular immunisation     

Neurogenic differentiation of murine and human adipose-derived stromal cells

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1-3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.     

Seasonal variation in the growth and nutrient content of yellow-birch replacement roots

Summary Modified air layers were established on lateral long roots of 9 yellow birch (Betula alleghniensis Britton) trees, and all replacement roots >. 5 cm long were harvested periodically during the 1971 and 1972 growing seasons. The first replacement roots grew 6 weeks after layer establishment. Root layers were inactive from 29 Oct. 71 to 5 May 72. Active root layers produced an average of 208 mg per tree during the first season and 198 mg per tree during the second season. Concentrations of N, P, K, Ca, Mg, Fe, Mn, Zn, and Al all varied within growing season, and average concentration of some elements—Ca in particular—varied between growing seasons. This technique shows promise for studying the nutrient status of root systems of forest trees.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.