Abstract
Simulation experiments were used to show the impact of varying extraction efficiency, aliquot proportion, and PCR efficiency on the heterozygote balance of a range of diploid and haploid cells. Reducing either parameters introduces variance. It is well-known that the variance in heterozygote balance increases as the amount of DNA is reduced. Surprisingly the distribution is in fact diamond shaped — the variance start to decrease at very low amounts of DNA. Simulations suggest that pristine diluted DNA is an acceptable approximation in validations to infer heterozygote balance. However, the difference in distribution of the variance between diploid and haploid cell types may, under some circumstances, need to be considered in statistical models. Finally, we exemplify how simulations can be used to predict the outcome of PCR for degraded samples. Visualizing the predicted DNA profile as an electropherogram can help to identify the best approach for sample processing.
Highlights
The typical forensic DNA analysis process consists of sample recovery, extraction, quantification, amplification, and capillary electrophoresis
We have shown that the distribution is diamond shaped
The variance starts to decrease at very low amounts of DNA (50 pg or less, depending on PCR efficiency, aliquot proportion, and extraction efficiency) and the distributions become multimodal rather than continuous
Summary
The typical forensic DNA analysis process consists of sample recovery, extraction, quantification, amplification, and capillary electrophoresis. Parameters for comparison between simulated dilutions and simulated crime stains were as follows: the original experiment was a 2-fold serial dilution of a NIST human DNA quantitation standard (SRM 2372A) as outlined by [18] In this simulation, the series was extended at the lower end with three more dilutions to produce a final range of 1.65 to 845 pg in the PCR reaction (equivalent to 0.25–128 diploid cells assuming 6.6 pg per cell). Exe = 0.30 and pcra = 0.35, emulating a realistic process where a relatively efficient extraction method is combined with a relatively high aliquot proportion.. 3. For each simulated sample pcrc = 30 − 35 PCR cycles with PCR efficiency pcre = 0.90, approximately in the middle of a previously reported range of 0.82 − 0.97 [10], was used. The R packages data.table6. and plyr7. were used for some calculations for performance reasons. ggplot28. was used to create figures
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