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Targeted S5 RNA sequencing assay for the identification and direct association of common body fluids with DNA donors in mixtures

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Abstract

The evidentiary value of DNA profiles varies depending upon the context in which the DNA was found. Linking a DNA profile to a particular cellular phenotype in mixtures may aid in assessing its evidentiary relevance and value. We report the development of two dual-function high-resolution messenger RNA (mRNA) sequencing assays that can each identify the presence of 6 body fluids/tissues (blood, semen, saliva, vaginal secretions, menstrual blood, skin) and, via coding region SNPs (cSNPs) present in the body fluid–specific mRNA transcripts, directly associate particular body fluids with their specific DNA donors in mixtures. The original blood, semen, and saliva (BSS) assay contains 23 cSNPs for blood, semen, and saliva, while the expanded 6F (all 6 fluids/tissues) assay encompasses the BSS assay and also contains 23 additional cSNPs for vaginal secretions, menstrual blood, and skin. Software tools were developed to infer the identity of the body fluids present as well as providing the corresponding cSNP genotypes. Concomitant genomic DNA assays (BSS-d and 6F-d), required to genotype the same cSNPs from persons of interest/inferred contributors to the body fluid mixture, were also developed. Body fluid specificity was demonstrated by the ability to identify the body fluid origin of single-source and two-fluid admixtures. The discriminatory power (European Caucasians) for all body fluids is 0.957–0.997, with linkage disequilibrium considered. Reciprocal body fluid admixtures (mixture pairs with the same two donors but reversed body fluid types) were used to demonstrate the ability to identify the body fluid source of origin as well as associate the donor of each of the two fluids.

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Data availability

The BFID-cSNP-BSS and 6F assays may be made available through Thermo Fisher Scientific as a community panel.

Code availability

Any associated analysis software will be made available if possible through Thermo Fisher Scientific.

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Acknowledgements

The authors would like to thank the anonymous donors who provided samples for this study.

Funding

This work was funded by the National Institute of Justice (NIJ), Office of Justice Programs, U.S. Department of Justice (Award No. 2014-DN-BX-K019), the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 285487 (EUROFORGEN-NoE), and a sponsored research agreement (SRA) with Thermo Fisher Scientific. The funding agencies had no role in study design, data analysis, and interpretation. The opinions, findings, and conclusions or recommendations are those of the authors and do not necessarily reflect those of the funding agencies.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Central Florida, P.O. Box 162367, Orlando, FL, 32816-2367, USA

    Erin Hanson & Jack Ballantyne

  2. National Center for Forensic Science, Orlando, FL, USA

    Erin Hanson & Jack Ballantyne

  3. Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland

    Guro Dørum, Manuel Zamborlin, Shouyu Wang, Mario Gysi, Sabrina Ingold & Cordula Haas

  4. Life Sciences/HID, Thermo Fisher Scientific, San Francisco, CA, USA

    Robert Lagace & Chantal Roth

Authors
  1. Erin Hanson
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  2. Guro Dørum
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  3. Manuel Zamborlin
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  4. Shouyu Wang
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  5. Mario Gysi
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  9. Cordula Haas
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  10. Jack Ballantyne
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Contributions

Experimental design and methodology: Erin Hanson, Cordula Haas, Guro Dørum, and Jack Ballantyne; primer design: Rob Lagace; experimentation and data analysis: Erin Hanson, Sabrina Ingold, Manuel Zamborlin, Shouyu Wang, Mario Gysi, Cordula Haas, and Jack Ballantyne; software/program analysis: Rob Lagace and Chantal Roth; statistical analysis: Guro Dørum; writing of manuscript: Erin Hanson, Jack Ballantyne, and Guro Dørum; reviewing and editing of manuscript: Cordula Haas, Guro Dørum, Sabrina Ingold, Manuel Zamborlin, Shouyu Wang, Mario Gysi, and Rob Lagace.

Corresponding author

Correspondence to Jack Ballantyne.

Ethics declarations

Ethics approval and consent to participate

All procedures performed in this study were in accordance with the ethical standards of the institutions and with the 1964 Helsinki Declaration and its later amendments. The study was approved by the University of Central Florida’s Institutional Review Board (IRB #SBE-14–10768) and by the local ethics commission (Kantonale Ethikkommision (KEK) Zurich; declaration of no objection no. 24–2015). Informed consent to participate in the study was obtained from each donor and included the use of samples for body fluid identification and SNP and DNA genotyping.

Competing interests

Authors Hanson, Zamborlin, Wang, Dørum, Haas, Ingold, and Ballantyne declare no competing interests. Authors Lagace and Roth are employed by Thermo Fisher Scientific who provided partial support for this work.

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Hanson, E., Dørum, G., Zamborlin, M. et al. Targeted S5 RNA sequencing assay for the identification and direct association of common body fluids with DNA donors in mixtures. Int J Legal Med 137, 13–32 (2023). https://doi.org/10.1007/s00414-022-02908-9

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