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Massively parallel sequencing analysis of nondegraded and degraded DNA mixtures using the ForenSeq™ system in combination with EuroForMix software

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Abstract

Massively parallel sequencing (MPS) technologies enable the simultaneous analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). MPS also enables the detection of alleles of the minor contributors in imbalanced DNA mixtures. In this study, 59 STRs (amelogenin, 27 autosomal STRs, 7 X-STRs, and 24 Y-STRs) and 94 identity-informative SNPs of 119 unrelated Taiwanese (50 men, 69 women) were sequenced using a commercial MPS kit. Forty-eight nondegraded and 44 highly degraded two-person artificial DNA mixtures with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, and 1:99) were analyzed to examine the performance of this system for detecting the alleles of the minor contributors in DNA mixtures. Likelihood ratios based on continuous model were calculated using the EuroForMix for DNA mixture interpretation. The STR and SNP genotypes of these 119 Taiwanese were obtained. Several sequence variants of STRs were observed. Using EuroForMix software based on the sequence data of autosomal STRs and autosomal SNPs, 97.9% (47/48) and 97.7% (42/43) of minor donors were accurately inferred among the successfully analyzed nondegraded and degraded DNA mixtures, respectively. In conclusion, combined with EuroForMix software, this commercial kit is effective for assignment of the minor contributors in nondegraded and degraded DNA mixtures.

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References

  1. Dixon LA, Murray CM, Archer EJ, Dobbins AE, Koumi P, Gill P (2005) Validation of a 21-locus autosomal SNP multiplex for forensic identification purposes. Forensic Sci Int 154:62–77

    Article  CAS  PubMed  Google Scholar 

  2. Hwa HL, Chang YY, Lee JC, Lin CY, Yin HY, Tseng LH, Su YN, Ko TM (2012) Fifteen non-CODIS autosomal short tandem repeat loci multiplex data from nine population groups living in Taiwan. Int J Legal Med 126:671–675

    Article  PubMed  Google Scholar 

  3. Hwa HL, Wu LS, Lin CY, Huang TY, Yin HI, Tseng LH, Lee JC (2016) Genotyping of 75 SNPs using arrays for individual identification in five population groups. Int J Legal Med 130:81–89

    Article  PubMed  Google Scholar 

  4. Pakstis AJ, Speed WC, Fang R, Hyland FC, Furtado MR, Kidd JR, Kidd KK (2010) SNPs for a universal individual identification panel. Hum Genet 127:315–324

    Article  PubMed  Google Scholar 

  5. Pakstis AJ, Haigh E, Cherni L, ElGaaied ABA, Barton A, Evsanaa B, Togtokh A, Brissenden J, Roscoe J, Bulbul O, Filoglu G, Gurkan C, Meiklejohn KA, Robertson JM, Li CX, Wei YL, Li H, Soundararajan U, Rajeevan H, Kidd JR, Kidd KK (2015) 52 additional reference population samples for the 55 AISNP panel. Forensic Sci Int Genet 19:269–271

    Article  CAS  PubMed  Google Scholar 

  6. Walsh S, Chaitanya L, Clarisse L, Wirken L, Draus-Barini J, Kovatsi L, Maeda H, Ishikawa T, Sijen T, de Knijff P, Branicki W, Liu F, Kayser M (2014) Developmental validation of the HIrisPlex system: DNA-based eye and hair colour prediction for forensic and anthropological usage. Forensic Sci Int Genet 9:150–161

    Article  CAS  PubMed  Google Scholar 

  7. Churchill JD, Schmedes SE, King JL, Budowle B (2016) Evaluation of the Illumina(®) beta version ForenSeq™ DNA signature prep kit for use in genetic profiling. Forensic Sci Int Genet 20:20–29

    Article  CAS  PubMed  Google Scholar 

  8. Silvia AL, Shugarts N, Smith J (2017) A preliminary assessment of the ForenSeq™ FGx system: next generation sequencing of an STR and SNP multiplex. Int J Legal Med 131:73–86

    Article  PubMed  Google Scholar 

  9. Rockenbauer E, Hansen S, Mikkelsen M, Børsting C, Morling N (2014) Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing. Forensic Sci Int Genet 8:68–72

    Article  CAS  PubMed  Google Scholar 

  10. Van Neste C, Vandewoestyne M, Van Criekinge W, Deforce D, Van Nieuwerburgh F (2014) My-forensic-loci-queries (MyFLq) framework for analysis of forensic STR data generated by massive parallel sequencing. Forensic Sci Int Genet 9:1–8

    Article  CAS  PubMed  Google Scholar 

  11. Børsting C, Morling N (2015) Next generation sequencing and its applications in forensic genetics. Forensic Sci Int Genet 18:78–89

    Article  CAS  PubMed  Google Scholar 

  12. Kim EH, Lee HY, Yang IS, Jung SE, Yang WI, Shin KJ (2016) Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons. Forensic Sci Int Genet 22:1–7

    Article  CAS  PubMed  Google Scholar 

  13. Van Neste C, Van Nieuwerburgh F, Van Hoofstat D, Deforce D (2012) Forensic STR analysis using massive parallel sequencing. Forensic Sci Int Genet 6:810–818

    Article  CAS  PubMed  Google Scholar 

  14. Warshauer DH, Davis CP, Holt C, Han Y, Walichiewicz P, Richardson T, Stephens K, Jager A, King J, Budowle B (2015) Massively parallel sequencing of forensically relevant single nucleotide polymorphisms using TruSeq™ forensic amplicon. Int J Legal Med 129:31–36

    Article  PubMed  Google Scholar 

  15. van der Gaag KJ, de Leeuw RH, Hoogenboom J, Patel J, Storts DR, Laros JFJ, de Knijff P (2016) Massively parallel sequencing of short tandem repeats-population data and mixture analysis results for the PowerSeq™ system. Forensic Sci Int Genet 24:86–96

    Article  CAS  PubMed  Google Scholar 

  16. Hussing C, Børsting C, Mogensen HS, Morling N (2015) Testing of the Illumina® ForenSeq™ kit. Forensic Sci Int genet Suppl. Ser 5:e449–e450

    Google Scholar 

  17. Xavier C, Parson W (2017) Evaluation of the Illumina ForenSeq™ DNA signature prep kit - MPS forensic application for the MiSeq FGx™ benchtop sequencer. Forensic Sci Int Genet 28:188–194

    Article  CAS  PubMed  Google Scholar 

  18. Jäger AC, Alvarez ML, Davis CP, Guzmán E, Han Y, Way L, Walichiewicz P, Silva D, Pham N, Caves G, Bruand J, Schlesinger F, Pond SJ, Varlaro J, Stephens KM, Holt CL (2017) Developmental validation of the MiSeq FGx forensic genomics system for targeted next generation sequencing in forensic DNA casework and database laboratories. Forensic Sci Int Genet 28:52–70

    Article  CAS  PubMed  Google Scholar 

  19. Bleka Ø, Storvik G, Gill P (2016) EuroForMix: an open source software based on a continuous model to evaluate STR DNA profiles from a mixture of contributors with artefacts. Forensic Sci Int Genet 21:35–44

    Article  CAS  PubMed  Google Scholar 

  20. Bleka Ø, Eduardoff M, Santos C, Phillips C, Parson W, Gill P (2017) Open source software EuroForMix can be used to analyze complex SNP mixtures. Forensic Sci Int Genet 31:105–110

    Article  CAS  PubMed  Google Scholar 

  21. Just RS, Irwin JA (2018) Use of the LUS in sequence allele designations to facilitate probabilistic genotyping of NGS-based STR typing results. Forensic Sci Int Genet 34:197–205

    Article  CAS  PubMed  Google Scholar 

  22. Hwa HL, Chung WC, Chen PL, Lin CP, Li HY, Yin HI, Lee JC (2018) A 1204-single nucleotide polymorphism and insertion-deletion polymorphism panel for massively parallel sequencing analysis of DNA mixtures. Forensic Sci Int Genet 32:94–101

    Article  CAS  PubMed  Google Scholar 

  23. Illumina, Inc. (2015) Illumina ForenSeq™ DNA Signature Prep Guide (Part # 15049528 Rev. D), http://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/forenseq/forenseq-dna-signature-prep-guide-15049528-d.pdf. accessed 13 November 2017

  24. Illumina, Inc. (2016) ForenSeq™ Universal Analysis Software Guide (15053876 v01), https://support.illumina.com/content/dam/illumina-support/documents/documentation/software_documentation/forenseq-universal-analysis-software/forenseq-universal-analysis-software-guide-15053876-01.pdf. accessed 13 November 2017

  25. Wendt FR, King JL, Novroski NM, Churchill JD, Ng J, Oldt RF, McCulloh KL, Weise JA, Smith DG, Kanthaswamy S, Budowle B (2017) Flanking region variation of ForenSeq™ DNA signature prep kit STR and SNP loci in Yavapai native Americans. Forensic Sci Int Genet 28:146–154

    Article  CAS  PubMed  Google Scholar 

  26. Fattorini P, Previderé C, Carboni I, Marrubini G, Sorçaburu-Cigliero S, Grignani P, Bertoglio B, Vatta P, Ricci U (2017) Performance of the ForenSeq™ DNA signature prep kit on highly degraded samples. Electrophoresis 38:1163–1174

    Article  CAS  PubMed  Google Scholar 

  27. Guo F, Yu J, Zhang L, Li J (2017) Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA signature prep kit on the MiSeq FGx™ forensic genomics system. Forensic Sci Int Genet 31:135–148

    Article  CAS  PubMed  Google Scholar 

  28. Gettings KB, Kiesler KM, Vallone PM (2015) Performance of a next generation sequencing SNP assay on degraded DNA. Forensic Sci Int Genet 19:1–9

    Article  CAS  PubMed  Google Scholar 

  29. Guo SW, Tompson EA (1992) Performing the exact test of Hardy–Weinberg proportion for multiple alleles. Biometrics 48:361–372

    Article  CAS  PubMed  Google Scholar 

  30. Slatkin M (1994) Linkage disequilibrium in growing and stable populations. Genetics 137:331–336

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Zaiontz C (2017) Cronbach’s Alpha. http://www.real-statistics.com/reliability/cronbachs-alpha/. accessed 13 November 2017

  32. Buckleton J, Curran J (2008) A discussion of the merits of random man not excluded and likelihood ratios. Forensic Sci Int Genet 2:343–348

    Article  PubMed  Google Scholar 

  33. Butler JM (2015) Introduction and issue summary: new trends in forensic genetics. Forensic Sci Int Genet 18:1–3

    Article  PubMed  Google Scholar 

  34. Grandell I, Samara R, Tillmar AO (2016) A SNP panel for identity and kinship testing using massive parallel sequencing. Int J Legal Med 130:905–914

    Article  PubMed  Google Scholar 

  35. Bland JM, Altman DG (1995) Multiple significance tests: the Bonferroni methods. BMJ 310:170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hill CR, Kline MC, Coble MD, Butler JM (2008) Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples. J Forensic Sci 53:73–80

    Article  CAS  PubMed  Google Scholar 

  37. Aponte RA, Gettings KB, Duewer DL, Coble MD, Vallone PM (2015) Sequence-based analysis of stutter at STR loci: characterization and utility. Forensic Sci Int Genet Suppl Ser 5:e456–e458

    Article  Google Scholar 

  38. Bieber FR, Buckleton JS, Budowle B, Butler JM, Coble MD (2016) Evaluation of forensic DNA mixture evidence: protocol for evaluation, interpretation, and statistical calculations using the combined probability of inclusion. BMC Genet 17:125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Just RS, Moreno LI, Smerick JB, Irwin JA (2017) Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens. Forensic Sci Int Genet 28:1–9

    Article  CAS  PubMed  Google Scholar 

  40. Zeng X, King JL, Stoljarova M, Warshauer DH, LaRue BL, Sajantila A, Patel J, Storts DR, Budowle B (2015) High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing. Forensic Sci Int Genet 16:38–47

    Article  CAS  PubMed  Google Scholar 

  41. Seo SB, King JL, Warshauer DH, Davis CP, Ge J, Budowle B (2013) Single nucleotide polymorphism typing with massively parallel sequencing for human identification. Int J Legal Med 127:1079–1086

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors thank the National Center for Genome Medicine at Academia Sinica, Taiwan, for performance of genome-wide analysis with Genome-Wide Human SNP Array 6.0. This Center was supported by grants from the National Core Facility Program for Biotechnology of National Science Council, Taiwan, R.O.C. We acknowledge Mr. Chun-Yen Lin and Ms. Tsun-Ying Huang for technical support on degraded DNA analysis. We thank Jia-Ching Hwa for manuscript typing and partial data calculation. Special thanks are given to the hundreds of individuals who volunteered to provide biological samples for allele frequency data studies.

Funding

This work was supported by the Ministry of Science and Technology, Taiwan, R.O.C. [grant numbers MOST 103-2320-B-002-023-MY2], and National Taiwan University Hospital Taiwan, R.O.C. [grant numbers NTUH103-S2398].

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Authors and Affiliations

  1. Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen Ai Rd., Taipei, 100, Taiwan

    Hsiao-Lin Hwa, Hsiang-I Yin, Tsui-Ting Lee & James Chun-I Lee

  2. Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung Shan S. Rd., Taipei, 100, Taiwan

    Hsiao-Lin Hwa & Ming-Yih Wu

  3. Department of Medical Genetics, National Taiwan University Hospital, No. 7 Chung Shan S. Rd., Taipei, 100, Taiwan

    Hsiao-Lin Hwa

  4. Yourgene Bioscience, No.376-5 Fuxing Rd., Shulin Dist., New Taipei City, 238, Taiwan

    Wan-Chia Chung & Chih-Peng Lin

  5. Genephile Bioscience Laboratory, No. 10, 1 F, Lin Sen South Road, Zhong Zheng District, Taipei, 100, Taiwan

    Tsang-Ming Ko

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  1. Hsiao-Lin Hwa
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Correspondence to James Chun-I Lee.

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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.

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Hwa, HL., Wu, MY., Chung, WC. et al. Massively parallel sequencing analysis of nondegraded and degraded DNA mixtures using the ForenSeq™ system in combination with EuroForMix software. Int J Legal Med 133, 25–37 (2019). https://doi.org/10.1007/s00414-018-1961-y

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  • DOI: https://doi.org/10.1007/s00414-018-1961-y

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