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Mixture deconvolution by massively parallel sequencing of microhaplotypes.
Int J Legal Med. 2019 05; 133(3):719-729.IJ

Abstract

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.

Authors+Show Affiliations

Metro Nashville Police Department Crime Laboratory, 400 Myatt Drive, Madison, TN, 37115, USA.The Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road NW, Washington, D.C., 20007, USA.The Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road NW, Washington, D.C., 20007, USA.The Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road NW, Washington, D.C., 20007, USA.The Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road NW, Washington, D.C., 20007, USA.Thermo Fisher Scientific, 180 Oyster Point Boulevard, San Francisco, CA, 94080, USA.Thermo Fisher Scientific, 180 Oyster Point Boulevard, San Francisco, CA, 94080, USA.Thermo Fisher Scientific, 180 Oyster Point Boulevard, San Francisco, CA, 94080, USA.Thermo Fisher Scientific, 180 Oyster Point Boulevard, San Francisco, CA, 94080, USA.Department of Genetics, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.The Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road NW, Washington, D.C., 20007, USA. podini@gwu.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30758713

Citation

Bennett, Lindsay, et al. "Mixture Deconvolution By Massively Parallel Sequencing of Microhaplotypes." International Journal of Legal Medicine, vol. 133, no. 3, 2019, pp. 719-729.
Bennett L, Oldoni F, Long K, et al. Mixture deconvolution by massively parallel sequencing of microhaplotypes. Int J Legal Med. 2019;133(3):719-729.
Bennett, L., Oldoni, F., Long, K., Cisana, S., Madella, K., Wootton, S., Chang, J., Hasegawa, R., Lagacé, R., Kidd, K. K., & Podini, D. (2019). Mixture deconvolution by massively parallel sequencing of microhaplotypes. International Journal of Legal Medicine, 133(3), 719-729. https://doi.org/10.1007/s00414-019-02010-7
Bennett L, et al. Mixture Deconvolution By Massively Parallel Sequencing of Microhaplotypes. Int J Legal Med. 2019;133(3):719-729. PubMed PMID: 30758713.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mixture deconvolution by massively parallel sequencing of microhaplotypes. AU - Bennett,Lindsay, AU - Oldoni,Fabio, AU - Long,Kelly, AU - Cisana,Selena, AU - Madella,Katrina, AU - Wootton,Sharon, AU - Chang,Joseph, AU - Hasegawa,Ryo, AU - Lagacé,Robert, AU - Kidd,Kenneth K, AU - Podini,Daniele, Y1 - 2019/02/13/ PY - 2018/01/29/received PY - 2019/01/23/accepted PY - 2019/2/14/pubmed PY - 2019/5/30/medline PY - 2019/2/14/entrez KW - Forensic DNA samples KW - Massively parallel sequencing (MPS) KW - Microhaplotype KW - Mixture deconvolution KW - Single-nucleotide polymorphism SP - 719 EP - 729 JF - International journal of legal medicine JO - Int J Legal Med VL - 133 IS - 3 N2 - 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. SN - 1437-1596 UR - https://www.unboundmedicine.com/medline/citation/30758713/Mixture_deconvolution_by_massively_parallel_sequencing_of_microhaplotypes_ L2 - https://dx.doi.org/10.1007/s00414-019-02010-7 DB - PRIME DP - Unbound Medicine ER -