International Journal of Legal Medicine

, Volume 132, Issue 2, pp 397–403 | Cite as

Rapidly mutating Y-STR analyses of compromised forensic samples

  • Rashed Alghafri
  • Irena Zupanič Pajnič
  • Tomaž Zupanc
  • Jože Balažic
  • Pankaj Shrivastava
Short Communication


Rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) were identified to improve differentiation of unrelated males and also to enable separating closely and distantly related males in human identity testing in forensic and other applications. RM-Yplex assay was developed as a single multiplex that is capable of simultaneously amplifying all currently known RM Y-STRs, and reproducibility and sensitivity testing were performed on reference samples. Additional analyses are necessary to test its suitability for analysing compromised forensic samples. For this purpose, we applied the RM-Yplex assay to approximately 70-year-old skeletons that were used as a model for poorly preserved, challenging forensic samples. We analysed 57 male skeletal remains (bones and teeth) from 55 skeletons excavated from the Second World War (WWII) mass graves in Slovenia. The RM-Yplex typing was successful in all 57 samples; there were 56% full profiles obtained, and in partial profiles, up to 7 locus drop-outs were observed and they appeared correlated with low DNA quantities and degradation of DNA obtained from WWII bone and tooth samples. The longest loci, DYS403S1b, DYS547, DYS627 and DYS526b, were the most often dropped-out RM Y-STRs. In spite of high frequency of drop-out events, the RM-Yplex typing was successful in all WWII samples, showing the possibility of successful amplification of at least half of the RM Y-STRs even from the most compromised samples analysed.


Rapidly mutating Y-STRs RM-Yplex Aged DNA Skeletal remains Second World War Compromised forensic samples 



The authors gratefully acknowledge Barbara Gornjak Pogorelc and Katja Vodopivec Mohorčič (Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana) for technical assistance. The authors like to thank the Governmental Commission on Concealed Mass Graves of the Republic of Slovenia for their support in excavations of the Second World War victims. This research did not receive any specific grant from founding agencies in the public, commercial or not-for-profit sectors.

Compliance with ethical standards

The research project was approved by the Medical Ethics Committee of the Republic of Slovenia (0120-156/2015-2; KME 122/06/15).

Supplementary material

414_2017_1600_MOESM1_ESM.xlsx (21 kb)
ESM 1 (XLSX 21 kb)


  1. 1.
    Adnan A, Ralf A, Rakha A, Kousouri N, Kayser M (2016) Improving empirical evidence on differentiating closely related men with RM Y-STRs: a comprehensive pedigree study from Pakistan. Forensic Sci Int Genet 25:45–51CrossRefPubMedGoogle Scholar
  2. 2.
    Alghafri R, Goodwin W, Ralf A, Kayser M, Hadi S (2015) A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs. Forensic Sci Int Genet 17:91–98CrossRefPubMedGoogle Scholar
  3. 3.
    Alonso A, Andelinović Š, Martin P (2001) DNA typing from skeletal remains: evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples. Croat Med J 42:260–266PubMedGoogle Scholar
  4. 4.
    Amory S, Huel R, Bilić A, Loreille O, Parsons TJ (2012) Automatable full demineralization DNA extraction procedure from degraded skeletal remains. Forensic Sci Int Genet 6:398–406CrossRefPubMedGoogle Scholar
  5. 5.
    Applied Biosystems (2003) Quantifiler Human DNA Quantification kit User Guide, Foster City (CA)Google Scholar
  6. 6.
    Baeta M, Núñez C, Cardoso S, Palencia-Madrid L, Herrasti L, Etxeberria F et al (2015) Digging up the recent Spanish memory: genetic identification of human remains from mass graves of the Spanish Civil War and posterior dictatorship. Forensic Sci Int Genet 19:272–279CrossRefPubMedGoogle Scholar
  7. 7.
    Ballantyne KN, Goedbloed M, Fang R, Schaap O, Lao O, Wollstein A et al (2010) Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications. Am J Hum Genet 87:341–353CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ballantyne KN, Keerl V, Wollstein A, Choi Y, Zuniga S, Ralf A et al (2012) A new future of forensic Y-chromosome analysis: rapidly mutating Y-STRs for differentiating male relatives and paternal lineages. Forensic Sci Int Genet 6:208–218CrossRefPubMedGoogle Scholar
  9. 9.
    Ballantyne KN, Ralf A, Aboukhalid R, Achakzai NM, Anjos MJ, Ayub Q et al (2014) Toward male individualization with rapidly mutating Y-chromosomal short tandem repeats. Hum Mutat 35:1021–1032CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Burger J, Hummel S, Hermann B, Henke W (1999) DNA preservation: a microsatellite-DNA study on ancient skeletal remains. Electrophoresis 20:1722–1728CrossRefPubMedGoogle Scholar
  11. 11.
    Caputo M, Irisarri M, Alechine E, Corach D (2013) A DNA extraction method of small quantities of bone for high-quality genotyping. Forensic Sci Int Genet 7:488–493CrossRefPubMedGoogle Scholar
  12. 12.
    Carracedo A, Bär W, Lincoln P, Mary W (2000) DNA commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing. Forensic Sci Int 110:79–85CrossRefPubMedGoogle Scholar
  13. 13.
    Chaitanya L, Zupanič Pajnič I, Walsh S, Balažic J, Zupanc T, Kayser M (2017) Bringing colour back after 70 years: predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system. Forensic Sci Int Genet 26:48–57CrossRefPubMedGoogle Scholar
  14. 14.
    D’Amato M, Ehrenreich L, Cloete K, Benjeddou M, Davison S (2010) Characterization of the highly discriminatory loci DYS449, DYS481, DYS518, DYS612, DYS626, DYS644 and DYS710. Forensic Sci Int Genet 4:104–110CrossRefPubMedGoogle Scholar
  15. 15.
    Fulton LT (2012) Setting up an ancient DNA laboratory. In: Shapiro B, Hofreiter M (eds) Ancient DNA - methods and Protocols. Humana Press Inc, New York, pp 1–11Google Scholar
  16. 16.
    Gill P, Whitaker J, Flaxman C, Brown N, Buckleton J (2000) An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Sci Int 112:17–40CrossRefPubMedGoogle Scholar
  17. 17.
    Keyser-Tracqui C, Ludes B (2005) Methods for the study of ancient DNA. In: Carracedo A (ed) Forensic DNA typing protocols. Humana Press Inc, New York, pp 253–264Google Scholar
  18. 18.
    Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–715CrossRefPubMedGoogle Scholar
  19. 19.
    Liu JY, Zhang C, Holt A, Lagace R, Harrold M, Dixon AB et al (2012) AutoMate Express forensic DNA extraction system for the extraction of genomic DNA from biological samples. J Forensic Sci 57:1022–1030CrossRefPubMedGoogle Scholar
  20. 20.
    Niederstätter H, Berger B, Kayser M, Parson W (2016) Differences in urbanization degree and consequences on the diversity of conventional vs. rapidly mutating Y-STRs in five municipalities from a small region of the Tyrolean Alps in Austria. Forensic Sci Int Genet 24:180–193CrossRefPubMedGoogle Scholar
  21. 21.
    Ossowski A, Kuś M, Kupiec T, Bykowska M, Zielińska G, Jasiński ME et al (2016) The Polish genetic database of victims of totalitarianisms. Forensic Sci Int 258:41–49CrossRefPubMedGoogle Scholar
  22. 22.
    Pääbo S, Poinar H, Serre D (2004) Genetic analyses from ancient DNA. Annu Rev Genet 38:645–679CrossRefPubMedGoogle Scholar
  23. 23.
    Parson W, Gusmao L, Hares DR, Irwin JA, Mayr WR, Morling N et al (2014) DNA Commission of the International Society for Forensic Genetics: revised and extended guidelines for mitochondrial DNA typing. Forensic Sci Int Genet 13:134–142CrossRefPubMedGoogle Scholar
  24. 24.
    Qiagen Companies (2014) EZ1 DNA Investigator Handbook, HildenGoogle Scholar
  25. 25.
    Rohland N, Hofreiter M (2007) Ancient DNA extraction from bones and teeth. Nat Protoc 2:1756–1762CrossRefPubMedGoogle Scholar
  26. 26.
    Tully G, Bär W, Brinkmann B, Carracedo A, Gill P, Morling N (2001) Considerations by the European DNA profiling (EDNAP) group on the working practices nomenclature and interpretation of mitochondrial DNA profiles. Forensic Sci Int 124:83–91CrossRefPubMedGoogle Scholar
  27. 27.
    Westen AA, Kraaijenbrink T, Clarisse L, Grol LJW, Willemse P, Zuniga SB et al (2015) Analysis of 36 Y-STR marker units including a concordance study among 2085 Dutch males. Forensic Sci Int Genet 14:174–181CrossRefPubMedGoogle Scholar
  28. 28.
    Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle B (1995) Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med 108:68–74CrossRefPubMedGoogle Scholar
  29. 29.
    Zupanič Pajnič I (2013a) Molekularnogenetska preiskava 300 let starih skeletov iz Auerspergove grobnice (Molecular genetic analyses of 300 years old skeletons from Auersperg tomb). Zdrav Vestn 82:796–808Google Scholar
  30. 30.
    Zupanič Pajnič I (2013b) Genetic identification of Second world war victim’s skeletal remains. Lap Lambert Academic Publishing, SaarbrückenGoogle Scholar
  31. 31.
    Zupanič Pajnič I (2016b) Extraction of DNA from human skeletal material. In: Goodwin W (ed) Forensic DNA typing protocols, methods in molecular biology, vol 1420. Springer Science&Business Media, LLC, New York. doi: 10.1007/978–1–4939-3597-0_7 Google Scholar
  32. 32.
    Zupanič Pajnič I, Zupanc T, Balažic J, Geršak ŽM, Stojković O, Skadrić I et al (2017) Prediction of autosomal STR typing success in ancient and Second World War bone samples. Forensic Sci Int Genet 27:17–26CrossRefPubMedGoogle Scholar
  33. 33.
    Zupanič-Pajnič I, Gornjak-Pogorelc B, Balažic J (2010) Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia. Int J Legal Med 124:307–317CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Zupanič-Pajnič I, Debska M, Gornjak-Pogorelc B, Vodopivec Mohorčič K, Balažic J et al (2016a) Highly efficient automated extraction of DNA from old and contemporary skeletal remains. J Forensic Legal Med 37:78–86CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.General Department of Forensic Sciences and Criminology, Dubai Police General Head QuartersDubaiUnited Arab Emirates
  2. 2.Institute of Forensic Medicine, Faculty of MedicineUniversity of LjubljanaLjubljanaSlovenia
  3. 3.DNA Fingerprinting Unit, State Forensic Science Laboratory, Govt. of Madhya PradeshSagarIndia

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