Short tandem repeats are the gold standard for human identification but are not informative for forensic DNA phenotyping (FDP). Single-nucleotide polymorphisms (SNPs) as genetic markers can be applied to both identification and FDP. The concept of DNA intelligence emerged with the potential for SNPs to infer biogeographical ancestry (BGA) and externally visible characteristics (EVCs), which together enable the FDP process. For more than a decade, the SNaPshot technique has been utilised to analyse identity and FDP-associated SNPs in forensic DNA analysis. SNaPshot is a single-base extension (SBE) assay with capillary electrophoresis as its detection system. This multiplexing technique offers the advantage of easy integration into operational forensic laboratories without the requirement for any additional equipment. Further, the SNP panels from SNaPshot assays can be incorporated into customised panels for massively parallel sequencing (MPS). Many SNaPshot assays are available for identity, BGA and EVC profiling with examples including the well-known SNPforID 52-plex identity assay, the SNPforID 34-plex BGA assay and the HIrisPlex EVC assay. This review lists the major forensically relevant SNaPshot assays for human DNA SNP analysis and can be used as a guide for selecting the appropriate assay for specific identity and FDP applications.
The ability to provide accurate DNA-based forensic intelligence requires analysis of multiple DNA markers to predict the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of biological evidence. Massively parallel sequencing (MPS) enables the analysis of hundreds of DNA markers in multiple samples simultaneously, increasing the value of the intelligence provided to forensic investigators while reducing the depletion of evidential material resulting from multiple analyses. The Precision ID Ancestry Panel (formerly the HID Ion AmpliSeq™ Ancestry Panel) (Thermo Fisher Scientific) (TFS)) consists of 165 autosomal SNPs selected to infer BGA. Forensic validation criteria were applied to 95 samples using this panel to assess sensitivity (1 ng-15 pg), reproducibility (inter- and intra-run variability) and effects of compromised and forensic casework type samples (artificially degraded and inhibited, mixed source and aged blood and bone samples). BGA prediction accuracy was assessed using samples from individuals who self-declared their ancestry as being from single populations of origin (n = 36) or from multiple populations of origin (n = 14). Sequencing was conducted on Ion 318™ chips (TFS) on the Ion PGM™ System (TFS). HID SNP Genotyper v4.3.1 software (TFS) was used to perform BGA predictions based on admixture proportions (continental level) and likelihood estimates (sub-population level). BGA prediction was accurate at DNA template amounts of 125pg and 30pg using 21 and 25 PCR cycles respectively. HID SNP Genotyper continental level BGA assignments were concordant with BGAs for self-declared East Asian, African, European and South Asian individuals. Compromised, mixed source and admixed samples, in addition to sub-population level prediction, requires more extensive analysis.
Forensic DNA-based intelligence, or forensic DNA phenotyping, utilises SNPs to infer the biogeographical ancestry and externally visible characteristics of the donor of evidential material. SNaPshot is a commonly employed forensic SNP genotyping technique, which is limited to multiplexes of 30-40 SNPs in a single reaction and prone to PCR contamination. Massively parallel sequencing has the ability to genotype hundreds of SNPs in multiple samples simultaneously by employing an oligonucleotide sample barcoding strategy. This study of the Illumina MiSeq massively parallel sequencing platform analysed 136 unique SNPs in 48 samples from SNaPshot PCR amplicons generated by five established forensic DNA phenotyping assays comprising the SNPforID 52-plex, SNPforID 34-plex, Eurasiaplex, Pacifiplex and IrisPlex. Approximately 3 GB of sequence data were generated from two MiSeq flow cells and profiles were obtained from just 0.25 ng of DNA. Compared with SNaPshot, an average 98% genotyping concordance was achieved. Our customised approach was successful in attaining SNP profiles from extremely degraded, inhibited, and compromised casework samples. Heterozygote imbalance and sequence coverage in negative controls highlight the need to establish baseline sequence coverage thresholds and refine allele frequency thresholds. This study demonstrates the potential of the MiSeq for forensic SNP analysis.
Massively parallel sequencing (MPS) is fast approaching operational use in forensic science, with the capability to analyse hundreds of DNA identity and DNA intelligence markers in multiple samples simultaneously. The ForenSeq™ DNA Signature Kit on MiSeq FGx™ (Illumina) workflow can provide profiles for autosomal short tandem repeats (STRs), X chromosome and Y chromosome STRs, identity single nucleotide polymorphisms (SNPs), biogeographical ancestry SNPs and phenotype (eye and hair colour) SNPs from a sample. The library preparation procedure involves a series of steps including target amplification, library purification and library normalisation. This study highlights the comparison between the manufacturer recommended magnetic bead normalisation and quantitative polymerase chain reaction (qPCR) methods. Furthermore, two qPCR chemistries, KAPA® (KAPA Biosystems) and NEBNext® (New England Bio Inc.), have also been compared. The qPCR outperformed the bead normalisation method, while the NEBNext® kit obtained higher genotype concordance than KAPA®. The study also established an MPS workflow that can be utilised in any operational forensic laboratory.
Single nucleotide polymorphisms (SNPs) have been widely used in forensics for prediction of identity, biogeographical ancestry (BGA) and externally visible characteristics (EVCs). Single base extension (SBE) assays, most notably SNaPshot® (Thermo Fisher Scientific), are commonly used for forensic SNP genotyping as they can be employed on standard instrumentation in forensic laboratories (e.g. capillary electrophoresis). High resolution melt (HRM) analysis is an alternative method and is a simple, fast, single tube assay for low throughput SNP typing. This study compares HRM and SNaPshot®. HRM produced reproducible and concordant genotypes at 500 pg, however, difficulties were encountered when genotyping SNPs with high GC content in flanking regions and differentiating variants of symmetrical SNPs. SNaPshot® was reproducible at 100 pg and is less dependent on SNP choice. HRM has a shorter processing time in comparison to SNaPshot®, avoids post PCR contamination risk and has potential as a screening tool for many forensic applications.
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