Forensic DNA methylation profiling from minimal traces: How low can we go?

Analysis of human DNA methylation (DNAm) can provide additional investigative leads in crime cases, e.g. the type of tissue or body fluid, the chronological age of an individual, and differentiation between identical twins. In contrast to the genetic profile, the DNAm level is not the same in every cell. At the single cell level, DNAm represents a binary event at a defined CpG site (methylated versus non-methylated). The DNAm level from a DNA extract however represents the average level of methylation of the CpG of interest of all molecules in the forensic sample. The variance of DNAm levels between replicates is often attributed to technological issues, i.e. degradation of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification failure. On the other hand, we show that stochastic variations can lead to gross fluctuation in the analysis of methylation levels in samples with low DNA levels. This stochasticity in DNAm results is relevant since low DNA amounts (1pg - 1ng) is rather the norm than the exception when analyzing forensic DNA samples. This study describes a conceptual analysis of DNAm profiling and its dependence on the amount of input DNA. We took a close look at the variation of DNAm analysis due to DNA input and its consequences for different DNAm-based forensic applications. As can be expected, the 95%-confidence interval of measured DNAm becomes narrower with increasing amounts of DNA. We compared this aspect for two different DNAm-based forensic applications: body fluid identification and chronological age determination. Our study shows that DNA amount should be well considered when using DNAm for forensic applications.