Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability

Abstract

A comparative study has been carried out, comparing two different methods to calculate activity level likelihood ratios (LRa) using Bayesian Networks. The first method uses the sub-source likelihood ratio (log10LRφ) as a 'quality indicator'. However, this has been criticised as introducing potential bias from population differences in allelic proportions. An alternative method has been introduced that is based upon the total RFU of a DNA profile that is adjusted, by multiplication, of the mixture proportion (Mx) which is calculated from quantitative probabilistic genotyping software (EuroForMix). Bayesian logistic regressions of direct transfer data showed that the two methods were comparable. Differences were attributed to sampling error, and small sample sizes of secondary transfer data. The Bayesian approach facilitates comparative studies by taking account of sampling error; it can easily be extended to compare different methods.

Transfer, persistence and recovery of DNA and mRNA vaginal mucosa markers after intimate and social contact with Bayesian network analysis for activity level reporting

“…23. The average RFU (RFU) per locus was multiplied by the dilution factor (d l ) for the PCR input (based on DNA concentration) and the mixture proportion (M X ) of the (POI), as described by Gill et al [34]:…”

“…Indirect transfer of DNA (s D ) was modelled from log normal distributions of penile and fingernail swabs; also modelled for both direct (t D ) and indirect (s D ) transfer for boxershorts, where there is no time dependency. Log-normal distributions were fitted to the data using the R package fitdistrplus using function lnorm [34]. The method is described in detail in Supplementary material 2.…”

“…The programming of these networks was carried out with R-code using the formulae described in Section 2.4. The 4000 bootstrapped samples from log normal distributions were combined with 4000 MCMC estimates from Bayesian logistic regression and were used in sensitivity analysis, described by quantiles (0.025 -0.975) to define the plausible range of LRs, as described by Gill et al [34].…”

Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability

“…The calculated is multiplied by the dilution factor to give the adjusted value. If the sample is a mixture, then it is also multiplied by the mixture proportion of the person of interest (POI) as described by [3] to give: …”

“…The notion of using average peak height of a DNA profile as a proxy measurement for DNA quantity was introduced by Tvedebrink et al [1, 2], along with a method to apportion quantities per contributor based on analysis of contributor-specific alleles. The method was improved by [3, 4, 5] who used probabilistic genotyping, EuroForMix [6], to determine the relative proportions of known and unknown contributors ( M x ), that constitute a mixture. The output was used to calculate likelihood ratios given activity level propositions based upon .…”