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Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems

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Abstract

Biological samples, including skeletal remains exposed to environmental insults for extended periods of time, exhibit increasing levels of DNA damage and fragmentation. Human forensic identification methods typically use a combination of mitochondrial (mt) DNA sequencing and short tandem repeat (STR) analysis, which target segments of DNA ranging from 80 to 500 base pairs (bps). Larger templates are often unavailable as skeletal samples age and the associated DNA degrades. Single-nucleotide polymorphism (SNP) loci target shorter templates and may serve as a solution to the problem. Recently developed assays for STR and SNP analysis using a massively parallel sequencing approach, such as the ForenSeq kit (Verogen, San Diego, CA), offer a means for generating results from degraded samples as they target templates down to 60 to 170 bps. We performed a modeling study that demonstrates that SNPs can increase the significance of an identification when analyzing DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. Observations from this study were then compared with human skeletal material results (n = 14, ninth to eighteenth centuries), which further demonstrated the utility of the ForenSeq kit for degraded samples. The robustness of the Promega PowerSeq™ Mito System was also tested with human skeletal remains (n = 70, ninth to eighteenth centuries), resulting in successful coverage of 99.29% of the mtDNA control region at 50× coverage or more. This was accompanied by modifications to a mainstream DNA extraction technique for skeletal remains that improved recovery of shorter templates.

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Acknowledgments

The authors would like to thank the following people for their input and guidance throughout the project: Jennifer McElhoe and Charity Holland (Holland Research laboratory at the Pennsylvania State University), Molly Rathbun (The Pennsylvania State University), Sylvain Amory, Stefan Prost, Rene Huel (ICMP), and Ana Bilic (ICMP). All sheared samples were prepared with the help of the Penn State Genomics Core Facility – University Park, PA.

Funding

This project was partially funded thanks to the Carol DeForest Grant from the Northeastern Association of Forensic Science, Illumina, and the National Institute of Justice (2015-DN-BX-K025).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Forensic Science Program, Pennsylvania State University, State College, PA, USA

    Elena I. Zavala, Swetha Rajagopal & Mitchell M. Holland

  2. Department of Forensic Science, John Jay College of Criminal Justice, New York, NY, USA

    Swetha Rajagopal

  3. Departments of Anthropology and Biology, Pennsylvania State University, State College, PA, USA

    George H. Perry

  4. University Department of Forensic Sciences, University of Split, Split, Croatia

    Ivana Kruzic & Željana Bašić

  5. International Commission on Missing Persons, The Hague, Netherlands

    Thomas J. Parsons

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  1. Elena I. Zavala
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  2. Swetha Rajagopal
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  4. Ivana Kruzic
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  5. Željana Bašić
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  7. Mitchell M. Holland
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Correspondence to Elena I. Zavala.

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Informed consent

Informed consent was obtained from all individual participants included in the study. Skeletal material was provided with permission from the University of Split.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The Ethical committee from the Medical School (University of Split) approved the research on skeletal remains (approval no. 45-1106 from 6 March 2006).

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Zavala, E.I., Rajagopal, S., Perry, G.H. et al. Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems. Int J Legal Med 133, 1369–1380 (2019). https://doi.org/10.1007/s00414-019-02110-4

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