New methods for quantification and analysis of quantitative real-time polymerase chain reaction data

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive gene quantitation method that has been widely used in the biological and biomedical fields. The currently used methods for PCR data analysis, including the threshold cycle (CT) method, linear and non-linear model fitting methods, all require subtracting background fluorescence. However, the removal of background fluorescence is usually inaccurate, and therefore can distort results. Here, we propose a new method, the taking-difference linear regression method, to overcome this limitation. Briefly, for each two consecutive PCR cycles, we subtracted the fluorescence in the former cycle from that in the later cycle, transforming the n cycle raw data into n-1 cycle data. Then linear regression was applied to the natural logarithm of the transformed data. Finally, amplification efficiencies and the initial DNA molecular numbers were calculated for each PCR run. To evaluate this new method, we compared it in terms of accuracy and precision with the original linear regression method with three background corrections, being the mean of cycles 1-3, the mean of cycles 3-7, and the minimum. Three criteria, including threshold identification, max R2, and max slope, were employed to search for target data points. Considering that PCR data are time series data, we also applied linear mixed models. Collectively, when the threshold identification criterion was applied and when the linear mixed model was adopted, the taking-difference linear regression method was superior as it gave an accurate estimation of initial DNA amount and a reasonable estimation of PCR amplification efficiencies. When the criteria of max R2 and max slope were used, the original linear regression method gave an accurate estimation of initial DNA amount. Overall, the taking-difference linear regression method avoids the error in subtracting an unknown background and thus it is theoretically more accurate and reliable. This method is easy to perform and the taking-difference strategy can be extended to all current methods for qPCR data analysis.^

Age -period -cohort analysis: An extension of Cox's lifetable regression with time -dependent covariates

It is well known that an identification problem exists in the analysis of age-period-cohort data because of the relationship among the three factors (date of birth + age at death = date of death). There are numerous suggestions about how to analyze the data. No one solution has been satisfactory. The purpose of this study is to provide another analytic method by extending the Cox's lifetable regression model with time-dependent covariates. The new approach contains the following features: (1) It is based on the conditional maximum likelihood procedure using a proportional hazard function described by Cox (1972), treating the age factor as the underlying hazard to estimate the parameters for the cohort and period factors. (2) The model is flexible so that both the cohort and period factors can be treated as dummy or continuous variables, and the parameter estimations can be obtained for numerous combinations of variables as in a regression analysis. (3) The model is applicable even when the time period is unequally spaced.^ Two specific models are considered to illustrate the new approach and applied to the U.S. prostate cancer data. We find that there are significant differences between all cohorts and there is a significant period effect for both whites and nonwhites. The underlying hazard increases exponentially with age indicating that old people have much higher risk than young people. A log transformation of relative risk shows that the prostate cancer risk declined in recent cohorts for both models. However, prostate cancer risk declined 5 cohorts (25 years) earlier for whites than for nonwhites under the period factor model (0 0 0 1 1 1 1). These latter results are similar to the previous study by Holford (1983).^ The new approach offers a general method to analyze the age-period-cohort data without using any arbitrary constraint in the model. ^

Longitudinal categorical data analysis: A continuous -time Markov chain approach

In this dissertation, we propose a continuous-time Markov chain model to examine the longitudinal data that have three categories in the outcome variable. The advantage of this model is that it permits a different number of measurements for each subject and the duration between two consecutive time points of measurements can be irregular. Using the maximum likelihood principle, we can estimate the transition probability between two time points. By using the information provided by the independent variables, this model can also estimate the transition probability for each subject. The Monte Carlo simulation method will be used to investigate the goodness of model fitting compared with that obtained from other models. A public health example will be used to demonstrate the application of this method. ^

Seagrass time-series analysis products, in Moreton Bay, Australia, with links to GeoTIFFs

The spatial and temporal dynamics of seagrasses have been well studied at the leaf to patch scales, however, the link to large spatial extent landscape and population dynamics is still unresolved in seagrass ecology. Traditional remote sensing approaches have lacked the temporal resolution and consistency to appropriately address this issue. This study uses two high temporal resolution time-series of thematic seagrass cover maps to examine the spatial and temporal dynamics of seagrass at both an inter- and intra-annual time scales, one of the first globally to do so at this scale. Previous work by the authors developed an object-based approach to map seagrass cover level distribution from a long term archive of Landsat TM and ETM+ images on the Eastern Banks (~200 km**2), Moreton Bay, Australia. In this work a range of trend and time-series analysis methods are demonstrated for a time-series of 23 annual maps from 1988 to 2010 and a time-series of 16 monthly maps during 2008-2010. Significant new insight was presented regarding the inter- and intra-annual dynamics of seagrass persistence over time, seagrass cover level variability, seagrass cover level trajectory, and change in area of seagrass and cover levels over time. Overall we found that there was no significant decline in total seagrass area on the Eastern Banks, but there was a significant decline in seagrass cover level condition. A case study of two smaller communities within the Eastern Banks that experienced a decline in both overall seagrass area and condition are examined in detail, highlighting possible differences in environmental and process drivers. We demonstrate how trend and time-series analysis enabled seagrass distribution to be appropriately assessed in context of its spatial and temporal history and provides the ability to not only quantify change, but also describe the type of change. We also demonstrate the potential use of time-series analysis products to investigate seagrass growth and decline as well as the processes that drive it. This study demonstrates clear benefits over traditional seagrass mapping and monitoring approaches, and provides a proof of concept for the use of trend and time-series analysis of remotely sensed seagrass products to benefit current endeavours in seagrass ecology.