An Automated Single Cell Separation Technique to Improve Mixture Deconvolution

Although the interpretation of mixed DNA samples is commonplace in forensic analyses, complex samples often present interpretational challenges. These complexities are introduced through a variety of environmental, biological, and process-related steps. Overly complex mixtures often cannot be interpreted with high confidence which can lead to inconclusive results, poor resolution between donors, and negatively affected likelihood ratios. This can occur in cases where samples collected from items that have been handled by numerous individuals or sexual assault evidence has significantly high epithelial cell to sperm cell ratios and low amounts of sperm.

This webinar will provide an evaluation of the DEPArray™, an instrument that can be used to identify, separate, and recover single cells or groups of cells prior to genetic analysis, thereby resolving challenges inherent to mixed biological evidence. Isolation of cells is performed using a single-use micro-fluidic cartridge containing an array of di-electrophoretic (DEP) cages (electrodes) that can be individually controlled permitting the capture and manipulation of single or groups of cells. Once captured, the cell(s) can then be routed to a recovery chamber by changing the electric field pattern and subsequently collected in a standard micro-centrifuge tube. The DEPArray™ has been used in the cancer research community since 2010 to primarily identify and isolate circulating tumor cells from large populations of white blood cells. Once this small population of target cells has been isolated from the non-target population, analyses such as PCR and whole genome sequencing can be used to characterize the sample in the absence of the “noise” created by non-target cells. This problem set largely mirrors those experienced in forensic DNA analyses, where samples are commonly composed of more than one donor of the same or differing cell types in differing quantities.

The webinar will focus on the findings of a study that incorporated the DEPArray™ into a standard forensic workflow—DNA extraction, PCR amplification using the PowerPlex Fusion 6c Human DNA amplification kit, (with manufacturer’s recommended number of cycles), and detection using the Applied Biosystems 3500xL Genetic Analyzer. The DEPArray™-mediated method was compared with traditional differential extraction; single source male profiles were obtained from 26 of 27 (96.2%) of DEPArray™ processed samples and only 9 of 28 (32.1%) differentially extracted samples. This study demonstrated that this workflow will lead to fewer mixtures and the ability to identify and isolate sperm cells that are present in exceeding low quantities. In addition, significant improvements were observed in the level of interpretable allelic responses from single, two, and three sperm samples. Additional benefits include the ability to avoid differential extractions and avoid the need for qPCR based quantification—where the DEPArray permits the user to physically count the number of cells, thereby accurately determining the true amount of DNA template in the sample.

The second part of this webinar will revisit the prospect of single cell analyses in forensic DNA analyses. The DEPArray provides a means to extract, purify, and recover single cells, without the loss experienced using other methods of cell separation. This, used in concert with the new expanded and more sensitive multiplex PCR kits, provides a new avenue by which to evaluate the ability to obtain interpretable, high quality data from single cells. We will provide preliminary results of a study that revisits single cell analyses in the context of forensic identification. Again, the DEPArray™ workflow was paired with the PowerPlex Fusion 6c human DNA amplification kit where PCR cycles were varied from 29 to 31 cycles. Overall data quality improved notably when increasing PCR from 29 to 30 cycles; however, less improvement and more volatility was introduced at 31 cycles. The average random match probabilities (RMP) for the 29, 30, and 31 cycle sets at 150RFU are 1 in 2.4x1018 ± 1.46x1019, 1 in 1.49x1025 ± 5.8x1025, and 1 in 1.83x1024 ± 8.09x1024, respectively, thereby demonstrating the feasibility of single cell analyses in the current forensic DNA pipeline.

Speakers

• Michael Marciano
• Victoria Williamson