The MASTiFF panel—a versatile multiple-allele SNP test for forensics


Forensic identification tests often need recourse to markers that can successfully type highly degraded DNA, and binary single nucleotide polymorphisms (SNPs) have become the variants of choice for such analyses because of their short amplified fragment lengths. The two main drawbacks of SNPs are their reduced power of discrimination per marker compared with mainstream forensic STRs and an inability to robustly detect mixed DNA—particularly using capillary electrophoresis genotyping systems such as SNaPshot™, where the dye signals are much more imbalanced than those of STR profiles. This study compiled a compact set of multiple-allele SNPs consisting of loci that had three or four nucleotide variants at the same site in order to address the lack of mixture detection capability with binary SNP tests, as well as improving levels of polymorphism per SNP by transitioning to a maximum of six or ten genotypes per locus. We report the development and optimisation of a SNaPshot-based forensic test comprising 27 tri-allelic and 2 tetra-allelic SNPs, which we named MASTiFF: a multiple-allele SNP test for forensics. Assessments of the MASTiFF panel’s levels of discrimination power in the five main population groups indicate random match probabilities ranging from 10−15 down to 10−20—improving the levels possible from an equivalent number of binary SNPs. The SNaPshot test was able to detect simple mixtures successfully with more than two alleles observed in 30% of SNPs. From allele frequency data, it is estimated that more than two alleles will be present in at least one MASTiFF SNP in 99.8% of two-person mixtures, making this panel an ideal supplementary test when SNPs are chosen for the analysis of degraded forensic DNA.

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Authors CP, MdlP, and MVL and the work of this study were supported by MAPA, “Multiple Allele Polymorphism Analysis” (BIO2016–78525-R), a research project funded by the Spanish Research State Agency (AEI), and co-financed with ERDF funds. MdlP was supported by a postdoctoral fellowship awarded by the Consellería de Cultura, Educación e Ordenación Universitaria, and the Consellería de Economía, Emprego e Industria from Xunta de Galicia (Modalidade A, ED481B 2017/088).

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Correspondence to C. Phillips.

Ethics declarations

The samples of the HGDP-CEPH population panel used in this study were collected with full informed consent from donors and ethical approval of the collection and distribution frameworks established by the Ethics Board of the Centre d’Etude du Polymorphisme Humain, Paris. Aspects of the ethical use of HGDP-CEPH DNA samples are discussed in detail by Fullerton and Lee, 2011 [45]. Although HGDP-CEPH donors are de-identified, we chose to only report summary allele frequencies from HGDP-CEPH Oceanian and American population groups. All other human variant data analysed in the study were obtained from the open access online data resources of 1000 Genomes, gnomAD, and Simons Foundation genome variation projects.

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The authors declare that they have no conflict of interest.

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Supplementary Figure S1

Example electropherograms from the two single base extension tests Auto 1 and Auto 2, analysing the standard control DNA 9947A (1 ng input). The rs-numbers corresponding to the internal codes TRI1, TRI2, etc. are outlined in Supplementary Tables 1/S2, columns B and C. (PNG 718 kb)

Supplementary Figure S2

Evaluation of the ancestry informativeness of the MASTiFF panel with a compact sample set from the five main continental population groups. (A) STRUCTURE cluster membership proportions for K:5, data merged data and plotted with CLUMPAK. (B) One out cross-validation success rates for ancestry assignments using Snipper. (C) Representation of the 3 coordinates of multi-dimensional scaling analysis. (D) Representation of the neighbour-joining tree analysis. (PNG 790 kb)


Supplementary Table S1 MASTiFF SNaPshot assay details. PCR primers are used in a single amplification reaction, but the single base extensions to detect the SNP alleles provides optimal profiles using POP-4™ polymer from two parallel reactions and separate electrophoretic separations denoted by clear (Auto 1) and grey (Auto 2) highlighted cells. Supplementary Table S2 Characteristics of electrophoretic artefacts observed in SNaPshot profiles and the peak of the closest SNP allele. IC: internal code. Supplementary Table S3 Allele frequency estimates of MASTiFF SNPs in five continental populations (AFR: 1000 Genomes African, EUR: 1000 Genomes European, EAS: 1000 Genomes East Asian, OCE: HGDP-CEPH Oceanian, AMR: HGDP-CEPH American). Ref.: reference allele (GRCh37/hg19) in 1000 Genomes Phase 3 database; Al.: alternative alleles 1, 2, 3 in alphabetic order. Supplementary Table S4 Genomic details and expected heterozygosity values for five continental populations (AFR, EUR, EAS, OCE, AMR) of the MASTiFF panel SNPs. SNPs highlighted in grey show high values in EUR which are much lower in other population groups. IC: internal code; Chr: chromosome; Ref. reference allele (GRCh37/hg19) in 1000 Genomes Phase 3 database; Al.1, 2, 3: alternative alleles in alphabetic order. Supplementary Table S5 Full population and group allele frequency estimates from the 1000 Genomes and gnomAD human variant databases. Supplementary Table S6A Genomic details of 29 MASTiFF SNPs. A4 alleles found at very low frequency in the gnomAD and TOPmed variant databases marked. Supplementary Table S6B GRCh37 genome build chromosome positions of MASTiFF (black) and 52plex (blue) SNPs. Boxes denote syntenic MASTiFF-52plex SNP pairs separated by less than 1 megabase (Mb). RA: reference allele; A2: allele-2, etc.; Chr: chromosome. (XLSX 93 kb)

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Phillips, C., Manzo, L., de la Puente, M. et al. The MASTiFF panel—a versatile multiple-allele SNP test for forensics. Int J Legal Med 134, 441–450 (2020).

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  • Single nucleotide polymorphisms (SNPs)
  • Multiple-allele SNPs
  • SNaPshot
  • Mixed DNA