Revisiting Single Cell Analysis in Forensic Science

Abstract

Background Forensic science has yet to take full advantage of single cell analysis. Its greatest benefit is the ability to alleviate the challenges associated with DNA mixture analysis, which, despite the emergence of probabilistic genotyping, remains a significant hurdle in forensic science. Many of the factors that cause complexity in mixture interpretation are absent in single cell analyses — multiple contributors and varied levels of contribution and allele masking. Recent technological innovations have been developed within the scientific community that mitigate the historical risks of single cell analysis in forensic casework. This study revisits single cell analyses in the context of forensic identification, introducing previously unseen depth to the characterization of data generated from single cells using a novel pipeline that includes recovery of single cells using the DEPArray™ NxT and amplification using the PowerPlex Fusion 6c kit with varied PCR cycles (29, 30, and 31); the resulting allelic signal was assessed using analytical thresholds of 10, 100 and 150RFU. Results The mean peak heights across the sample sets generally increased as cycle number increased, 75.0 ± 85.3, 147.1 ± 172.6, and 226.1 ± 298.2 RFU, for 29, 30, and 31 cycles, respectively. The average proportion of allele/locus dropout was most significantly impacted by changes in the detection threshold, whereas increases f PCR cycle number had less impact. Overall data quality improved notably when increasing PCR from 29 to 30 cycles; however, less improvement and more volatility introduced at 31 cycles. The average random match probabilities (RMP) for the 29, 30, and 31 cycle sets at 150RFU are 1 in 2.4 × 1018 ± 1.46 × 1019, 1 in 1.49 × 1025 ± 5.8 × 1025, and 1 in 1.83 × 1024 ± 8.09 × 1024, respectively. Conclusions This study introduces and optimizes an analytical pipeline to generate ample high quality data from single cells that can be used in forensic analyses, thus removing the need for complex mixture interpretation. An analysis of the impact of PCR cycles on single cells revealed a significant overall improvement in the amount and quality of interpretable data when increasing from 29 to 30 cycles, while no such improvement was noted when increasing to 31 cycles.

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

“…Notably, the median average peak height rendered by PP3—that is, low proteinase K and 0.5X PBS—is 667 RFU, which is consistent with the median peak heights obtained with single epithelial cells collected in the absence of PBS [17]. The peak heights obtained from PP1 and PP2 are similar to those obtained when using an unrelated, though analogous, treatment (i.e., LysePrep extraction and a 31 cycle PowerPlex Fusion 6c amplification) [19] showing that, in general, forensically relevant single‐cell pipelines are readily implemented using a variety of treatments and improved results can be obtained by systematically evaluating the effect of each component on EPG results.…”

“…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.…”

“…In addition, decreasing the proteinase K concentration had no detrimental effects. Since barriers to implementation of single‐cell technologies into crime laboratory environments will be driven by successful single‐cell EPG procurement, reasonable reagent costs and the development of sound interpretation techniques, this work supplements existing literature by demonstrating most scEPGs render good quality profiles when the individual system components are well‐joined and compatible [16,19–21,44,45]…”

“…Single‐cell adaptations to meet forensic requirements were described as early as 1997 [16], where the authors summarized that of the 226 micro‐dissected cells, 114 resulted in a full profile, and the allele dropout rate was a manageable 39%. More recently, semi‐automated procedures have been used to garner single‐cell data in a mechanized fashion [18–21]. The myriad ways single‐cell analysis can be implemented in the forensic laboratory can be divided into three broad categories: (a) manual micromanipulation techniques that rely on significant analyst engagement and include pico‐pipetting, laser micro‐dissection, and other micromanipulators; (b) semi‐automated methods that require the analyst to review and select cells for processing, though collection is managed by the instrumentation through microfluidics [19,20]; and (c) fully automated methods that do not rely on significant labor, and use micro‐ or nano‐liters of reagents [22].…”