Towards developing forensically relevant single-cell pipelines by incorporating direct-to-PCR extraction: compatibility, signal quality, and allele detection

Abstract

Current analysis of forensic DNA stains relies on the probabilistic interpretation of bulk-processed samples that represent mixed profiles consisting of an unknown number of potentially partial representations of each contributor. Single-cell methods, in contrast, offer a solution to the forensic DNA mixture problem by incorporating a step that separates cells before extraction. A forensically relevant single-cell pipeline relies on efficient direct-to-PCR extractions that are compatible with standard downstream forensic reagents. Here we demonstrate the feasibility of implementing single-cell pipelines into the forensic process by exploring four metrics of electropherogram (EPG) signal quality-i.e., allele detection rates, peak heights, peak height ratios, and peak height balance across low-to high-molecular-weight short tandem repeat (STR) markers-obtained with four direct-to-PCR extraction treatments and a common post-PCR laboratory procedure. Each treatment was used to extract DNA from 102 single buccal cells, whereupon the amplification reagents were immediately added to the tube and the DNA was amplified/injected using post-PCR conditions known to elicit a limit of detection (LoD) of one DNA molecule. The results show that most cells, regardless of extraction treatment, rendered EPGs with at least a 50% true positive allele detection rate and that allele drop-out was not cell independent. Statistical tests demonstrated that extraction treatments significantly impacted all metrics of EPG quality, where the Arcturus® PicoPure™ extraction method resulted in the lowest median allele drop-out rate, highest median average peak height, highest median average peak height ratio, and least negative median values of EPG sloping for GlobalFiler™ STR loci amplified at half volume. We, therefore, conclude the feasibility of implementing single-cell pipelines for casework purposes and demonstrate that inferential systems assuming cell independence will not be appropriate in the probabilistic interpretation of a collection of single-cell EPGs.

High‐quality data from a forensically relevant single‐cell pipeline enabled by low PBS and proteinase K concentrations

“…In contrast to bulk pipelines that produce a single EPG per crime‐scene sample, the single‐cell pipeline produces n scEPGs per sample—one for every cell collected. Despite the sensitive post‐PCR conditions and the absence of fractionation from the pipeline, scEPGs are known to vary in their quality [16,17,19], wherein some display peaks at every allele position across all loci, while others exhibit partial or full profile drop‐out, as illustrated in Figure 2A. As described in section 2.2, the cell is collected into a 0.2 mL amplification tube in proprietary DEPArray™ Buffer.…”

“…The extraction mix was prepared by adding reconstitution buffer into one vial of lyophilized proteinase K enzyme [34]. Since preliminary experiments using the manufacturer's recommendations showed lower than expected signal quality when compared with those acquired from micromanipulation methods [17], four collection/extraction reagent modifications were prepared and tested on single leukocytes collected from the DEPArray™ instrument. As depicted in Table 1, three concentrations of PBS—that is, 1X (10 mM), 0.5X (0.5 mM), 0.25X (0.25 mM)—were trialed, as were two proteinase K concentrations.…”

“…The frequency of allele detection was performed on a per cell basis since previous work demonstrated that there is inconsistency in the quality of the signal of EPGs generated from distinct cells and that a single likelihood of allele drop-out does not effectively characterize those data [17]. Specifically, rates of allele drop-out were determined by counting the number of heterozygous alleles crossing the AT of 30 RFU and dividing that value by the total number of expected heterozygous alleles based on the known genotype.…”

“…Single‐cell laboratory systems, for example, require direct‐to‐PCR reagents that are compatible with downstream PCR ones. In bulk mixture samples, adverse effects of reagent compatibility are less obvious, but in the single‐cell regime small PCR inefficiencies can have a significant impact on profile qualities [17].…”

Precision DNA Mixture Interpretation with Single-Cell Profiling

“…Thus, technology and marker specific ADO and ADI models may be the better options. In addition, the independence of the ADO and/or ADI events among the cells may not be assumed with real samples [69]. Therefore, more sophisticated ADO and ADI models that incorporate the underlying dependence between the cells may be better refracting the actual scenarios.…”