Abstract
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 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, varied levels of contribution, and allele masking. 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. 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 in PCR cycle number had less impact. Overall data quality improved notably when increasing PCR from 29 to 30 cycles, less improvement and more volatility was introduced at 31 cycles. The average random match probabilities 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. This demonstrates the current power of single cell analysis in removing the need for complex mixture analysis.
Highlights
Forensic science has yet to take full advantage of single cell analysis
Strategies exist to further optimize the analysis of single cells, the most notable of which is increasing the number of PCR cycles; whereby increases in cycle number theoretically correspond to increases in total allelic product and decrease the occurrence of allele and locus dropout
This study provides a comprehensive characterization of the behavior of single cells in a forensic pipeline and, in doing so, strengthens support for the use of single cell analyses. Implementation of this pipeline will alleviate many of the complexities associated with mixture analysis, including but not limited to, simplifying data interpretation, improving overall laboratory efficiency, and increasing the effectiveness of downstream statistical analyses
Summary
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 remains a significant hurdle in forensic science. The most significant challenge facing the success of single cell analysis, and low template analysis in general, is the combination of the limited amount of template DNA (approximately 6.6 pg) and the sensitivity of the analytical methods This leads to stochastic events that manifest as severe heterozygote imbalance and allele or locus dropout. Strategies exist to further optimize the analysis of single cells, the most notable of which is increasing the number of PCR cycles; whereby increases in cycle number theoretically correspond to increases in total allelic product and decrease the occurrence of allele and locus dropout The utility of this approach has been demonstrated by several studies[16,17,18,19,20,21], where low template DNA (< 100 pg) has been shown to provide adequate genetic information when adjusting the PCR cycle n umber[21]. A balance must be struck between the amount of interpretable DNA data obtained and the number of PCR cycles
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