Total Alleles Lost
3. Trends in Electropherogram Data from Compromised DNA Samples 1 Methods
3.2.5. Comparison Between Methods of Compromise 1 RFU Signal Decrease
3.2.5.3. Comparing Regression Lines to DI Values
Using the information provided by the equations for the trends in fragment size and peak height, the accuracy of the DI value given by the Quantifiler® Trio kit can be evaluated. The DI value is calculated using Equation 1 in Section 1.4. The SA amplicon size detected by Quantifiler® Trio is 80 base pairs, and the LA amplicon size target is 214 base pairs [32].
Using the exponential equations for the data, the values of f(x=80 bp) and f(x=214 bp) can be calculated. This will give the RFU values for amplicons at the SA and LA regions, which are approximately proportional to the concentration of undegraded template DNA with the concentrations [5-7, 56], and if the DI value provided by Quantifiler® Trio is accurate, then the following equation should be correct:
!(#$)
! &'( = DI value (Equation 5)
Figures 9 through 12, seen below, plot the DI value given for each sample from the Quantifiler® Trio kit versus the calculated DI using the exponential regression line equations. If the hypothesis that ! &'(!(#$) = DI value were true, the slope of the fitted linear trend line for the plot of each set of data would be equal to one.
Figure 9. Scatter plot data showing the Quantifiler® Trio DI Value vs. ! &'(!(#$)
calculated from the exponential regression lines of each dye channel for uncompromised DNA data.
Figure 10. Scatter plot data showing the Quantifiler® Trio DI Value vs. ! &'(!(#$)
calculated from the exponential regression lines of each dye channel for each sonication level of sonicated DNA data.
y = 0.8361x + 0.2148
0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 f( 80 )/ f( 21 4) Quantifiler Trio DI ValueUncompromised DNA
y = 1.8599x + 0.6972
0 2 4 6 8 10 12 14 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 f( 80 )/ f( 21 4) Quantifiler Trio DI ValueSonicated DNA
Figure 11. Scatter plot data showing the Quantifiler® Trio DI Value vs. !(#$)
! &'( calculated from the exponential regression lines of each dye channel for all exposure levels of UV-irradiated DNA.
Figure 12. Scatter plot data showing the Quantifiler® Trio DI Value vs. ! &'(!(#$)
calculated from the exponential regression lines of each dye channel for all
y = 0.0378x + 3.5718
0 1 2 3 4 5 6 0 10 20 30 40 50 f( 80 )/ f( 21 4) Quantifiler Trio DI ValueUV-Damaged DNA
y = 0.0029x + 2.0771
1.9 1.95 2 2.05 2.1 2.15 2.2 2.25 2.3 2.35 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 f( 80 )/ f( 21 4) Quantifiler Trio DI ValueEnzyme-Degraded DNA
The trend line for uncompromised DNA is the closest to the expected results, with a slope of 0.8361. The slope of the linear regression line for the sonicated DNA samples is equal to 1.8599, meaning that the DI value calculated by plugging the SA and LA values into the exponential regression is almost twice that of the DI value provided by Quantifiler® Trio kit. The slopes of the lines for samples exposed to UV light and enzymatic degradation are both very low, meaning the Quantifiler® Trio DI value is much larger than the value calculated by the exponential regression equations. The data from enzyme degraded DNA does not appear to be best fit by a linear regression line, as seen in Figure 7.
In all samples evaluated, the DI value did not have 1:1 correlation with the theoretical DI value calculated using the exponential regression lines fit to the data. Based on the fact that the slope of the uncompromised data, seen in Figure 7, does not equal the expected value of 1, qPCR-based DI values should be taken as an indication or approximation of degradation rather than a means to accurately predict the sloping effect in the electropherogram. Further, these data suggest that if the qPCR-based DI value is large, larger targets may be warranted, as severely degraded samples exhibited relatively low peak heights, even at small molecular weight loci.
4. Conclusion
Interpretation of uncompromised LTDNA profile data presents an inherent challenge due to the increased influence of stochastic effects. These stochastic effects can produce a configuration of peaks in the electropherogram that is different from the genotype of the DNA's donor due to missing information and peak height resolution. Software interpretation of profiles containing allelic drop- out, either due to stochastic effects or compromise, can result in false negatives leading to an exclusion of an individual because alleles were not observed in the profile. False negatives are perhaps inevitable for genotyping in the case of compromised LTDNA or those subject to stochastic loss. The unavoidable error made during genotyping suggests that a detailed understanding of the DNA signal for a variety of sample types is necessary.
Understanding the complex relationship between RFU signal and molecular weight in STR profiles is the key to being able to predict behavior patterns in compromised DNA samples. Only by thoroughly examining and comparing the trends in electropherogram data in artificially degraded DNA in controlled laboratory settings is it possible for improvement and standardization in STR typing procedures for highly degraded, LTDNA. Some distinctive trends in the compromised DNA data were observed, which not only suggests that impacts on RFU signal may be compromise-dependent, but is also promising for the possibility of classifying typical patterns in RFU signal decrease for a given method of compromise. With further characterization studies into the effects on
RFU signal decrease resulting from compromised LTDNA samples, it is possible to classify and predict typical behavior of profile data, thus minimizing the potential for Type II errors in DNA interpretation.
The dynamic model for allelic loss potential during the silica DNA extraction procedure shows the possibility for detrimental loss of genetic material by stochastic variation. In the future, studies can use this model to isolate specific steps in the procedure which are causing the most loss, and either modify the protocol to increase DNA yield or develop chemical reagents and technology for extraction of LTDNA with improved efficiencies.
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