Mixture deconvolution by massively parallel sequencing of microhaplotypes
Short tandem repeat polymorphisms (STRs) are the standard markers for forensic human identification. STRs are highly polymorphic loci analyzed using a direct PCR-to-CE (capillary electrophoresis) approach. However, STRs have limitations particularly when dealing with complex mixtures. These include slippage of the polymerase during amplification causing stutter fragments that can be indistinguishable from minor contributor alleles, preferential amplification of shorter alleles, and limited number of loci that can be effectively co-amplified with CE. Massively parallel sequencing (MPS), by enabling a higher level of multiplexing and actual sequencing of the DNA, provides forensic practitioners an increased power of discrimination offered by the sequence of STR alleles and access to new sequence-based markers. Microhaplotypes (i.e., microhaps or MHs) are emerging multi-allelic loci of two or more SNPs within < 300 bp that are highly polymorphic, have alleles all of the same length, and do not generate stutter fragments. The growing number of loci described in the literature along with initial mixture investigations supports the potential for microhaps to aid in mixture interpretation and the purpose of this study was to demonstrate that practically. A panel of 36 microhaplotypes, selected from a set of over 130 loci, was tested with the Ion S5™ MPS platform (Thermo Fisher Scientific) on single-source samples, synthetic two-to-six person mixtures at different concentrations/contributor ratios, and on crime scene-like samples. The panel was tested both in multiplex with STRs and SNPs and individually. The analysis of single-source samples showed that the allele coverage ratio across all loci was 0.88 ± 0.08 which is in line with the peak height ratio of STR alleles in CE. In mixture studies, results showed that the input DNA can be much higher than with conventional CE, without the risk of oversaturating the detection system, enabling an increased sensitivity for the minor contributor in imbalanced mixtures with abundant amounts of DNA. Furthermore, the absence of stutter fragments simplifies the interpretation. On casework-like samples, MPS of MHs enabled the detection of a higher number of alleles from minor donors than MPS and CE of STRs. These results demonstrated that MPS of microhaplotypes can complement STRs and enhance human identification practices when dealing with complex imbalanced mixtures.
KeywordsMicrohaplotype Single-nucleotide polymorphism Massively parallel sequencing (MPS) Mixture deconvolution Forensic DNA samples
The authors thank Dr. Moses S. Schanfield for providing the DNA samples, collected and extracted between 1993 and 2003, and used in this study.
This study was in part supported by National Institute of Justice through grants No. 2017-DN-BX-0164 awarded to Daniele Podini and No. 2015-DN-BX-K023 awarded to Kenneth K. Kidd, and by the Swiss National Science Foundation through grant No. 2017-P2LAP3_174742 awarded to Fabio Oldoni.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- 8.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 SJK, 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. https://doi.org/10.1016/j.fsigen.2017.01.011 CrossRefPubMedGoogle Scholar
- 9.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. https://doi.org/10.1016/j.fsigen.2016.05.016 CrossRefPubMedGoogle Scholar
- 11.Børsting C, Fordyce SL, Olofsson J, Mogensen HS, Morling N (2014) Evaluation of the Ion TorrentTM HID SNP 169-plex: a SNP typing assay developed for human identification by second generation sequencing. Forensic Sci Int Genet 12:144–154. https://doi.org/10.1016/j.fsigen.2014.06.004 CrossRefPubMedGoogle Scholar
- 12.Ambers AD, Churchill JD, King JL, Stoljarova M, Gill-King H, Assidi M, Abu-Elmagd M, Buhmeida A, Budowle B, Budowle B (2016) More comprehensive forensic genetic marker analyses for accurate human remains identification using massively parallel DNA sequencing. BMC Genomics 17:750. https://doi.org/10.1186/s12864-016-3087-2 CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Wendt FR, Warshauer DH, Zeng X, Churchill JD, Novroski NMM, Song B, King JL, LaRue BL, Budowle B (2016) Massively parallel sequencing of 68 insertion/deletion markers identifies novel microhaplotypes for utility in human identity testing. Forensic Sci Int Genet 25:198–209. https://doi.org/10.1016/j.fsigen.2016.09.005 CrossRefPubMedGoogle Scholar
- 14.Brown H, Thompson R, Murphy G, Peters D, La Rue B, King J, Montgomery AH, Carroll M, Baus J, Sinha S, Wendt FR, Song B, Chakraborty R, Budowle B, Sinha SK (2017) Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21. Forensic Sci Int Genet 29:80–99. https://doi.org/10.1016/j.fsigen.2017.03.017 CrossRefPubMedGoogle Scholar
- 23.Bulbul O, Pakstis AJ, Soundararajan U, Gurkan C, Brissenden JE, Roscoe JM, Evsanaa B, Togtokh A, Paschou P, Grigorenko EL, Gurwitz D, Wootton S, Lagace R, Chang J, Speed WC, Kidd KK (2017) Ancestry inference of 96 population samples using microhaplotypes. Int J Legal Med 132:703–711. https://doi.org/10.1007/s00414-017-1748-6 CrossRefPubMedPubMedCentralGoogle Scholar
- 25.van der Gaag KJ, de Leeuw RH, Laros JFJ, den Dunnen JT, de Knijff P (2018) Short hypervariable microhaplotypes: a novel set of very short high discriminating power loci without stutter artefacts. Forensic Sci Int Genet 35:169–175. https://doi.org/10.1016/j.fsigen.2018.05.008 CrossRefPubMedGoogle Scholar
- 28.Zhu J, Lv M, Zhou N, Chen D, Jiang Y, Wang L, He W, Peng D, Li Z, Qu S, Wang Y, Wang H, Luo H, An G, Liang W, Zhang L (2018) Genotyping polymorphic microhaplotype markers through the Illumina® MiSeq platform for forensics. Forensic Sci Int Genet 39:1–7. https://doi.org/10.1016/j.fsigen.2018.11.005 CrossRefPubMedGoogle Scholar
- 29.Chen P, Zhu W, Tong F, Pu Y, Yu Y, Huang S, Li Z, Zhang L, Liang W, Chen F (2018) Identifying novel microhaplotypes for ancestry inference. Int J Legal Med. https://doi.org/10.1007/s00414-018-1881-x
- 30.Butler JM, Decker AE, Kline MC, Reid TM, Vallone PM (2007) New autosomal and Y-chromosome STR loci: characterization and potential uses. In: 18th Int. Symp. Hum. Identification, Hollywood, CA, PromegaGoogle Scholar
- 33.Phillips C, Fang R, Ballard D, Fondevila M, Harrison C, Hyland F, Musgrave-Brown E, Proff C, Ramos-Luis E, Sobrino B, Carracedo A, Furtado MR, Court DS, Schneider PM (2007) Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel. Forensic Sci Int Genet 1:180–185. https://doi.org/10.1016/j.fsigen.2007.02.007 CrossRefPubMedGoogle Scholar
- 34.Holland MM, Fisher DL, Lee DA, Bryson CK, Weedn VW (1993) Short tandem repeat loci: application to forensic and human remains identification. In: Pena SDJ, Chakraborty R, Epplen JT, Jeffreys AJ (eds) DNA Fingerprinting State Sci. Birkhäuser Basel, Basel, pp 267–274. https://doi.org/10.1007/978-3-0348-8583-6_24 CrossRefGoogle Scholar
- 41.Wang DY, Gopinath S, Lagacé RE, Norona W, Hennessy LK, Short ML, Mulero JJ (2015) Developmental validation of the GlobalFiler® express PCR amplification kit: a 6-dye multiplex assay for the direct amplification of reference samples. Forensic Sci Int Genet 19:148–155. https://doi.org/10.1016/j.fsigen.2015.07.013 CrossRefPubMedGoogle Scholar
- 42.Budowle B, Giusti AM, Waye JS, Baechtel FS, Fourney RM, Adams DE, Presley LA, Deadman HA, Monson KL (1991) Fixed-bin analysis for statistical evaluation of continuous distributions of allelic data from VNTR loci, for use in forensic comparisons. Am J Hum Genet 48:841–855 https://www.ncbi.nlm.nih.gov/pubmed/1673286 PubMedPubMedCentralGoogle Scholar
- 43.T.I.H. 3 Consortium, Altshuler DM, Gibbs RA, Peltonen L, Altshuler DM, Gibbs RA, Peltonen L, Dermitzakis E, Schaffner SF, Yu F, Peltonen L, Dermitzakis E, Bonnen PE, Altshuler DM, Gibbs RA, de Bakker PIW (Co-leader), Deloukas P (Co-leader), Gabriel SB, Gwilliam R, Hunt S, Inouye M (Co-leader), Jia X, Palotie A, Parkin M (Co-leader), Whittaker P, Yu F (Leader), Chang K, Hawes A, Lewis LR, Ren Y, Wheeler D, Gibbs RA, Marie Muzny D, Barnes C, Darvishi K, Hurles M (Co-leader), Korn JM, Kristiansson K, Lee C, McCarroll SA (Co-leader), Nemesh J, Dermitzakis E, Keinan A (Leader), Montgomery SB, Pollack S, Price AL, Soranzo N, Bonnen PE, Gibbs RA, Gonzaga-Jauregui C, Keinan A, Price AL, Yu F (Leader), Anttila V, Brodeur W, Daly MJ, Leslie S, McVean G, Moutsianas L, Nguyen H, Schaffner SF (Leader), Zhang Q, Ghori MJR, McGinnis R (Co-leader), McLaren W, Pollack S, Price AL (Co-leader), Schaffner SF (Co-leader), Takeuchi F, Grossman SR, Shlyakhter I, Hostetter EB, Sabeti PC (Leader), Adebamowo CA, MW Foster, Gordon DR, Licinio J, Manca MC, Marshall PA, Matsuda I, Ngare D, Wang VO, Reddy D, Rotimi CN, Royal CD, Sharp RR, Zeng C, Brooks LD, McEwen JE (2010) Integrating common and rare genetic variation in diverse human populations. Nature 467:52. https://doi.org/10.1038/nature09298