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    Towards developing forensically relevant single-cell pipelines by incorporating direct-to-PCR extraction: compatibility, signal quality, and allele detection

    Sheth, Nidhi and Swaminathan, Harish and Gonzalez, Amanda G. and Duffy, Ken R. and Grgicak, Catherine (2021) Towards developing forensically relevant single-cell pipelines by incorporating direct-to-PCR extraction: compatibility, signal quality, and allele detection. International Journal of Legal Medicine, 135. pp. 727-738. ISSN 1437-1596

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    Current analysis of forensic DNA stains relies on the probabilistic interpretation of bulk-processed samples that represent mixed profiles consisting of an unknown number of potentially partial representations of each contributor. Single-cell methods, in contrast, offer a solution to the forensic DNA mixture problem by incorporating a step that separates cells before extraction. A forensically relevant single-cell pipeline relies on efficient direct-to-PCR extractions that are compatible with standard down- stream forensic reagents. Here we demonstrate the feasibility of implementing single-cell pipelines into the forensic process by exploring four metrics of electropherogram (EPG) signal quality—i.e., allele detection rates, peak heights, peak height ratios, and peak height balance across low- to high-molecular-weight short tandem repeat (STR) markers—obtained with four direct-to-PCR extraction treatments and a common post-PCR laboratory procedure. Each treatment was used to extract DNA from 102 single buccal cells, whereupon the amplification reagents were immediately added to the tube and the DNA was amplified/injected using post-PCR conditions known to elicit a limit of detection (LoD) of one DNA molecule. The results show that most cells, regardless of extraction treatment, rendered EPGs with at least a 50% true positive allele detection rate and that allele drop-out was not cell independent. Statistical tests demonstrated that extraction treatments significantly impacted all metrics of EPG quality, where the Arcturus® PicoPure™ extraction method resulted in the lowest median allele drop-out rate, highest median average peak height, highest median average peak height ratio, and least negative median values of EPG sloping for GlobalFiler™ STR loci amplified at half volume. We, therefore, conclude the feasibility of implementing single-cell pipelines for casework purposes and demonstrate that inferential systems assuming cell independence will not be appropriate in the probabilistic interpretation of a collection of single-cell EPGs.

    Item Type: Article
    Keywords: Forensic DNA; Human identification; Single-cell forensic DNA; Direct-to-PCR; Forensic DNA Mixtures;
    Academic Unit: Faculty of Science and Engineering > Research Institutes > Hamilton Institute
    Item ID: 15187
    Identification Number:
    Depositing User: Dr Ken Duffy
    Date Deposited: 06 Jan 2022 15:37
    Journal or Publication Title: International Journal of Legal Medicine
    Publisher: Springer
    Refereed: Yes
    Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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