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

Abstract

Interpreting forensic DNA signal is arduous since the total intensity is a cacophony of signal from noise, artifact, and allele from an unknown number of contributors (NOC).An alternate to traditional bulk-processing pipelines is a single-cell one, where the sample is collected, and each cell is sequestered resulting in n single-source, singlecell EPGs (scEPG) that must be interpreted using applicable strategies. As with all forensic DNA interpretation strategies, high quality electropherograms are required; thus, to enhance the credibility of single-cell forensics, it is necessary to produce an efficient direct-to-PCR treatment that is compatible with prevailing downstream laboratory processes.We incorporated the semi-automated micro-fluidic DEPArray™ technology into the single-cell laboratory and optimized its implementation by testing the effects of four laboratory treatments on single-cell profiles. We focused on testing effects of phosphate buffer saline (PBS) since it is an important reagent that mitigates cell rupture but is also a PCR inhibitor. Specifically, we explored the effect of decreasing PBS concentrations on five electropherogram-quality metrics from 241 leukocytes: profile drop-out, allele drop-out, allele peak heights, peak height ratios, and scEPG sloping. In an effort to improve reagent use, we also assessed two concentrations of proteinase K. The results indicate that decreasing PBS concentrations to 0.5X or 0.25X improves scEPG quality, while modest modifications to proteinase K concentrations did not significantly impact it. We, therefore, conclude that a lower than recommended proteinase K concentration coupled with a lower than recommended PBS concentration results in enhanced scEPGs within the semi-automated single-cell pipeline.

A systematic approach to improve downstream single‐cell analysis for the DEPArray™ technology

“…However, this approach is worth discussing, since high stutter ratios with large variance are already associated with low-template samples [51], which challenges the identification of the true allelic signal of unknown sources in casework. Thus, we agreed to define drop-in as an extraneous peak that is exceeding the analytical threshold and did not fall within a stutter or allele position, as described by Sheth et al [25] Even though increased stutter peaks, i.e. above 15% have been described…”

“…• Single-cell profiling at 12.5 μL show prominent stutters but of acceptable frequency. [6,9,25]. Nonetheless, STR profiling strategies for single cells differ among research groups in terms of preferred STR kits, PCR volumes or cycles, and threshold settings (Table 1).…”

“…Possible cell loss during cell routing was considered to be reduced by adjusting the routing speed to 900 ms, since a reduction should give the target cell, especially larger ones, additional time to follow the changes in di-electrophoretic pattern that forces the trapped cell to the next field minima position. Volume reduction was performed according to the manufacturer's recommendations using the VRNxT instrument (Menarini Silicon Biosystems).This minimized cell loss due to not using an additional pipetting step and ensured a final volume of 2 μL in each tube for DNA extraction by mitigating user-specific variable results[25]. The cell suspension was pelleted using a regular swinging bucket centrifuge.…”

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

“…Within this study, although a 2% v/v SDS solution exhibiting precipitation during extraction was used, it did not seem to negatively affect DNA purification. Due to the purification step within the extraction, also no inhibitory effects on the PCR caused by SDS or PBS could be determined in any of the samples, as already stated in other publications [65, 66]. Considering solely potential inhibitory effects when using other extraction or direct amplification methods, different moistening agents than SDS or PBS, for example, nonionic surfactants (i.e., Triton‐X‐100), might be advantageous.…”

Recent advances in forensic biology and forensic DNA typing: INTERPOL review 2019–2022

“…A number of approaches have been taken and advances made in DNA mixture interpretation [ 259 ]. These include probabilistic genotyping software [ 15 ], using genetic markers beyond traditional autosomal STR typing [ 260 ], or separating contributor cells and performing single-cell analysis [ [261] , [262] , [263] , [264] , [265] , [266] ].…”