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Forensic performance of two insertion–deletion marker assays

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Abstract

Improving the amplification and analysis of highly degraded DNA extracts has been a longstanding area of research in forensic genetics. One of the most promising recent developments in analysis of degraded DNA is the availability of short, biallelic insertion–deletion length polymorphisms (InDels) in highly multiplexed assays. InDels share many of the favourable characteristics of single-nucleotide polymorphisms (SNPs) that make them ideal markers for analysis of degraded DNA, including: analysis in short amplicon size ranges, high multiplexing capability and low mutation rates. In addition, as length-based polymorphisms, InDels can be analysed with the same simple dye-labelled PCR primer methods as standard forensic short tandem repeats. Separation and detection of fluorescently dye-labelled PCR products by capillary electrophoresis eliminate the multiple step protocols required by SNP typing with single-base extension assays and provide a closer relationship between the input DNA and the profile peak height ratios. Therefore InDel genotyping represents an effective new approach for human identification that adds informative new loci to the existing battery of forensic markers. To assess the utility of InDels for forensic analysis, we characterised population variation with two InDel identification assays: the 30-plex Qiagen DIPplex panel and a 38-plex panel developed by Pereira et al. in 2009 [1]. Allele frequencies were generated for the 68 markers in US African American, Caucasian, East Asian and Hispanic samples. We made a thorough assessment of the individual and combined performance of the InDel sets, as well as characterising profile artifacts and other issues related to the routine use of these newly developed forensic assays based on artificially degraded DNA and mixed source samples.

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Acknowledgments

The authors wish to thank Margaret Kline and Becky Hill at NIST as well as Anke Prochnow at Qiagen. The work of MF at NIST has been supported by the Fundacion Barrie de la Maza postgrade grant program (2010). MVL was supported by funding from Xunta de Galicia INCITE 09 208163PR and Ministerio de Educación y Ciencia BIO2006-06178. The support by funding from the Fundacion Marcelino Botin to the Institute of Legal Medicine of the USC. CS was partially supported through a PhD grant (SFRH/BD/75627/2010) awarded by the Portuguese Foundation for Science and Technology (FCT) and co-financed by the European Social Fund (Human Potential Thematic Operational Program). The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an Associate Laboratory of the Portuguese Ministry of Education and Science and is partially supported by FCT. RP holds an FCT fellowship (SFRH/BPD/81986/2011). LG is supported by a grant from CAPES/Brazil.

Disclaimer

This work was funded in part through an interagency agreement between the National Institute of Justice and the NIST Office of Law Enforcement Standards as well as funding from the FBI Biometric Center of Excellence: ‘Forensic DNA Typing as a Biometric Tool’. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Justice. Commercial equipment, instruments, and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.

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Authors and Affiliations

  1. Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Galicia, Spain

    M. Fondevila, C. Phillips, C. Santos, A. Carracedo & M. V. Lareu

  2. IPATIMUP—Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal

    R. Pereira & L. Gusmão

  3. Laboratório de Genética Humana e Médica, Instituto de Ciências Biológicas, Universidade Federeal do Pará, Belém, Brazil

    L. Gusmão

  4. U.S. National Institute of Standards and Technology, Biochemical Science Division, Gaithersburg, MD, USA

    M. Fondevila, J. M. Butler & P. M. Vallone

Authors
  1. M. Fondevila
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  2. C. Phillips
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  3. C. Santos
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  4. R. Pereira
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  5. L. Gusmão
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  6. A. Carracedo
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  7. J. M. Butler
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  8. M. V. Lareu
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  9. P. M. Vallone
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Corresponding author

Correspondence to M. Fondevila.

Electronic supplementary materials

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Supplementary Fig. S1

Covaris AFA-treated DNA analysis. Four samples were 3.4 ng/μL and treated with: 1 100 cycles/burst, 5 min, 1-mL container; 2 1,000 cycles, 20 min, 1 mL; 3 1,000 cycles, 6 min, 100-μL container; 4 1,000 cycles, 20 min, 100 μL. The last conditions (sample 4) were chosen for artificial fragmentation PCR analysis. (JPEG 34 kb)

High-resolution image (TIFF 94 kb)

Supplementary Fig. S2

Individual PHR distribution charts for each artificial mixture arranged in similar format to the graphs if Fig. 2, with the addition of control heterozygote means, lower/upper quartile boxes and extreme values shown as whiskers. This allowed counts of mixture sample PHRs outside both these normal ranges, strongly indicating the presence of a mixture. (PDF 157 kb)

Supplementary Table S1

Summary genomic details and dye label combinations of component loci of DIPplex and 38plex InDel assays [1, 15]. (DOCX 20 kb)

Supplementary Table S2

Sequencing primers used to characterise four DIPplex InDels showing mobility shift (D84, D99) or imbalanced signal alleles (D83, D97). (DOCX 11 kb)

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Fondevila, M., Phillips, C., Santos, C. et al. Forensic performance of two insertion–deletion marker assays. Int J Legal Med 126, 725–737 (2012). https://doi.org/10.1007/s00414-012-0721-7

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  • DOI: https://doi.org/10.1007/s00414-012-0721-7

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