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

• NCJ Number: 307547
• Journal: Journal of Forensic Sciences Volume: 67 Issue: 2 Dated: 2022 Pages: 697-706
• Author(s): Nidhi Sheth; Ken R. Duffy; Catherine M. Grgicak
• Date Published: 2022
• Length: 10 pages
• Grant Number(s): 2020-R2-CX-0032; 2018-DU-BX-0185
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: Resolving the Forensic DNA Mixture Problem through the Development of End-to-End Single-Cell Pipelines; Selectively analyzing and interpreting DNA from multiple donors with a full Single-Cell strategy

Annotation

The authors of this study of the semi-automated micro-fluidic DEPArray™ technology conclude 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.

Abstract

The authors 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; they 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. 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. 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, single-cell 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. The authors 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, the authors 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, the authors also assessed two concentrations of proteinase K. (Published Abstract Provided)