Shotgun DNA sequencing for human identification: Dynamic SNP selection and likelihood ratio calculations accounting for errors.

Shotgun sequencing is a DNA analysis method that potentially determines the nucleotide sequence of every DNA fragment in a sample, unlike PCR-based genotyping methods that is widely used in forensic genetics and targets predefined short tandem repeats (STRs) or predefined single nucleotide polymorphisms (SNPs). Shotgun DNA sequencing is particularly useful for highly degraded low-quality DNA samples, such as ancient samples or those from crime scenes. Here, we developed a statistical model for human identification using shotgun sequencing data and developed formulas for calculating the evidential weight as a likelihood ratio (LR). The model uses a dynamic set of binary SNP loci and takes the error rate from shotgun sequencing into consideration in a probabilistic manner. To our knowledge, the method is the first to make this possible. Results from replicated shotgun sequencing of buccal swabs (high-quality samples) and hair samples (low-quality samples) were arranged in a genotype-call confusion matrix to estimate the calling error probability by maximum likelihood and Bayesian inference. Different genotype quality filters may be applied to account for genotyping errors. An error probability of zero resulted in the commonly used LR formula for the weight of evidence. Error probabilities above zero reduced the LR contribution of matching genotypes and increased the LR in the case of a mismatch between the genotypes of the trace and the person of interest. In the latter scenario, the LR increased from zero (occurring when the error probability was zero) to low positive values, which allow for the possibility that the mismatch may be due to genotyping errors. We developed an open-source R package, wgsLR, which implements the method, including estimation of the calling error probability and calculation of LR values. The R package includes all formulas used in this paper and the functionalities to generate the formulas.

Identification of individuals from low template blood samples using whole transcriptome shotgun sequencing.

Biological trace samples consisting of very few cells pose a challenge to conventional forensic genetic DNA analysis. RNA may be an alternative to DNA when handling low template samples. Whereas each cell only contains two copies of an autosomal DNA segment, the transcriptome retains much of the genomic variation replicated in abundant RNA fragments. In this study, we describe the development of a prototype RNA-based SNP selection set for forensic human identification from low template samples (50 pg gDNA). Whole blood from a subset of the Danish population (41 individuals) and blood stains subjected to degradation at room temperature for up to two weeks were analysed by whole transcriptome shotgun sequencing. Concordance was determined by DNA genotyping with the Infinium Omni5-4 SNP chip. In the 100 protein-coding genes with the most reads, 5214 bi-allelic SNPs with gnomAD minor allele frequencies > 0.1 in the African/African American, East Asian, and (non-Finnish) European populations were identified. Of these, 24 SNPs in 21 genes passed screening in whole blood and degraded blood stains, with a resulting mean match probability of 4.5 ∙ 10-9. Additionally, ancestry informative SNPs and SNPs in genes useful for body fluid identification were identified in the transcriptome. Consequently, shotgun sequencing of RNA from low template samples may be used for a vast host of forensic genetics purposes, including simultaneous human and body fluid identification, leading to direct donor identification in the identified body fluid.

Collaborative exercise: analysis of age estimation using a QIAGEN protocol and the PyroMark Q48 platform.

Human age estimation from trace samples may give important leads early in a police investigation by contributing to the description of the perpetrator. Several molecular biomarkers are available for the estimation of chronological age, and currently, DNA methylation patterns are the most promising. In this study, a QIAGEN age protocol for age estimation was tested by five forensic genetic laboratories. The assay comprised bisulfite treatment of the extracted DNA, amplification of five CpG loci (in the genes of ELOVL2, C1orf132, TRIM59, KLF14, and FHL2), and sequencing of the amplicons using the PyroMark Q48 platform. Blood samples from 49 individuals with ages ranging from 18 to 64 years as well as negative and methylation controls were analyzed. An existing age estimation model was applied to display a mean absolute deviation of 3.62 years within the reference data set. Key points: Age determination as an intelligence tool during investigations can be a powerful tool in forensic genetics.In this study, five laboratories ran 49 samples and obtained a mean absolute deviation of 3.62 years.Five markers were analyzed on a PyroMark Q48 platform.

Cleaning protocols in forensic genetic laboratories.

It is pivotal to avoid cross-sample contamination in forensic genetic laboratories and optimal cleaning protocols for the removal of DNA are essential. A survey was performed, and ten forensic genetic laboratories shared their cleaning protocols in pre-PCR and post-PCR laboratories. The cleaning frequencies on different surface areas were somewhat similar, whereas none of the laboratories used the same cleaning reagents. Therefore, the efficiencies of the cleaning protocol utilised were tested and compared. The results showed that freshly made household bleach and Virkon® removed all amplifiable DNA from the surfaces, whereas DNA AWAY™ and the disinfection reagents ethanol, isopropanol, and ChemGene HLD4L did not.

Longitudinal changes and variation in human DNA methylation analysed with the Illumina MethylationEPIC BeadChip assay and their implications on forensic age prediction.

DNA methylation, a pivotal epigenetic modification, plays a crucial role in regulating gene expression and is known to undergo dynamic changes with age. The present study investigated epigenome-wide methylation profiles in 64 individuals over two time points, 15 years apart, using the Illumina EPIC850k arrays. A mixed-effects model identified 2821 age-associated differentially methylated CpG positions (aDMPs) with a median rate of change of 0.18% per year, consistent with a 10-15% change during a human lifespan. Significant variation in the baseline DNA methylation levels between individuals of similar ages as well as inconsistent direction of change with time across individuals were observed for all the aDMPs. Twenty-three of the 2821 aDMPs were previously incorporated into forensic age prediction models. These markers displayed larger changes in DNA methylation with age compared to all the aDMPs and less variation among individuals. Nevertheless, the forensic aDMPs also showed inter-individual variations in the direction of DNA methylation changes. Only cg16867657 in ELOVL2 exhibited a uniform direction of the age-related change among the investigated individuals, which supports the current knowledge that CpG sites in ELOVL2 are the best markers for age prediction.

Prediction of chronological age and its applications in forensic casework: methods, current practices, and future perspectives.

Estimating an individual's age can be relevant in several areas primarily related to the clinical and forensic fields. In the latter, estimation of an individual's chronological age from biological material left by the perpetrator at a crime scene may provide helpful information for police investigation. Estimation of age is also beneficial in immigration cases, where age can affect the person's protection status under the law, or in disaster victim identification to narrow the list of potential missing persons. In the last decade, research has focused on establishing new approaches for age prediction in the forensic field. From the first forensic age estimations based on morphological inspections of macroscopic changes in bone and teeth, the focus has shifted to molecular methods for age estimation. These methods allow the use of samples from human biological material that does not contain morphological age features and can, in theory, be investigated in traces containing only small amounts of biological material. Molecular methods involving DNA analyses are the primary choice and estimation of DNA methylation levels at specific sites in the genome is the most promising tool. This review aims to provide an overview of the status of forensic age prediction using molecular methods, with particular focus in DNA methylation. The frequent challenges that impact forensic age prediction model development will be addressed, together with the importance of validation efforts within the forensic community.

DNA quality evaluation of formalin-fixed paraffin-embedded heart tissue for DNA methylation array analysis.

Archived formalin-fixed and paraffin-embedded (FFPE) heart tissue from autopsied individuals represents an important resource for investigating the DNA methylation of heart tissue of deceased individuals. The DNA quality of FFPE tissue from autopsies may be decreased, affecting the DNA methylation measurements. Therefore, inexpensive screening methods for estimating DNA quality are valuable. We investigated the correlation between the DNA quality of archived FFPE heart tissue examined with the Illumina Infinium HD FFPE QC assay (Infinium QC) and Thermo Fisher's Quantifiler Trio DNA Quantification kit (QuantifilerTrio), respectively, and the amount of usable DNA methylation data as measured by the probe detection rate (probe DR) obtained with the Illumina Infinium MethylationEPIC array. We observed a high correlation (r2 = 0.75; p < 10-11) between the QuantifilerTrio degradation index, DI, and the amount of usable DNA methylation data analysed with SeSAMe, whereas a much weaker correlation was observed between the Infinium QC and SeSAMe probe DR (r2 = 0.17; p < 0.05). Based on the results, QuantifilerTrio DI seems to predict the proportion of usable DNA methylation data analysed with the Illumina Infinium MethylationEPIC array and SeSAMe by a linear model: SeSAMe probe DR = 0.80-log10(DI) × 0.25.

Reproducibility of the Infinium methylationEPIC BeadChip assay using low DNA amounts.

The Infinium MethylationEPIC BeadChip (EPIC) is a reliable method for measuring the DNA methylation of more than 850,000 CpG positions. In clinical and forensic settings, it is critical to be able to work with low DNA amounts without risking reduced reproducibility. We evaluated the EPIC for a range of DNA amounts using two-fold serial dilutions investigated on two different days. While the ß-value distributions were generally unaffected by decreasing DNA amounts, the median squared Pearson's correlation coefficient (R2) of between-days ß-value comparisons decreased from 0.994 (500 ng DNA) to 0.957 (16 ng DNA). The median standard deviation of the ß-values was 0.005 and up to 0.017 (median of medians: 0.014) for ß-values around 0.6-0.7. With decreasing amounts of DNA from 500 ng to 16 ng, the percentage of probes with standard deviations ≤ 0.1 decreased from 99.9% to 99.4%. This study showed that high reproducibility results are obtained with DNA amounts in the range 125-500 ng DNA, while DNA amounts equal to 63 ng or below gave less reproducible results.

Altered gene expression levels of genes related to muscle function in adults with cerebral palsy.

Cerebral palsy (CP) is the most common cause of movement disorders in children. Next generation sequencing (NGS) studies have previously shown that expression levels are fundamentally different in children with CP compared to typically developing (TD). However, given that children are in full development, we might expect gene expression levels to change once maturity is reached. Therefore, the main purpose of this study was to investigate gene expression levels of 93 target genes in adults with CP using NGS on muscle biopsies of the gastrocnemius, taken from 22 participants (n = 12 adults with CP; n = 10 TD adults). Subsequently, we carried out NGS of the mitochondrial genome to identify mtDNA variants, and additionally we studied the mitochondrial content using transmission electron microscopy images of the gastrocnemius muscle. Finally, we compared systemic ion levels between TD adults and adults with CP. Differential gene expression levels were found in genes involved in muscle contraction (MYH1 and MYBPC2), mitochondrial function kATP5J, CYCS and NDUFB6), calcium handling (CAMK2B and ATP2A), metabolism (LPL), muscle signaling (MYC, CREB1, ACVR2B, LMNA and TRIM54), and ECM (TNC). There was no statistical significant difference between CP and TD for mtDNA variant frequencies and mitochondrial content. The ion levels of Ca2+, Na+ and K+ were statistically significantly reduced while the Cl- levels were significant increased in adults with CP compared to TD adults. These results highlight that most transcriptional differences are related to muscle function in adults with CP and that mitochondrial function might be altered but not mitochondrial content.

The transcriptome of hand eczema assessed by tape stripping.

BACKGROUND: No biomarkers have been identified that can classify subtypes of hand eczema (HE). Although skin biopsies represent the gold standard for investigations of the skin, the invasive technique is not favorable when investigating skin from sensitive areas. Recent advances in the use of skin-tape strips for molecular investigations enable noninvasive investigations of HE. OBJECTIVE: By using whole transcriptome sequencing (WTS), the molecular profile of HE according to different localizations on the hands, etiologies, and clinical/morphological subtypes was investigated. METHODS: Thirty adult, Danish HE patients, 12 with and 18 without concurrent atopic dermatitis (AD), as well as 16 controls were included. Tape strip samples were collected from lesional, nonlesional, and healthy skin. Total RNA was extracted and WTS was performed. RESULTS: The largest molecular difference of HE patients with and without AD was found in nonlesional skin areas and included a downregulation of CXCL8 for HE patients without AD. Differences between allergic and irritant contact dermatitis included epidermal biomarkers such as EPHA1. CONCLUSION: Skin tape strip samples could be used to assess the gene expression profile of HE on different localizations of the hands. The skin tape strip method identified new molecular markers that showed promising result for the identification of HE subtypes.

Differential Methylation in the GSTT1 Regulatory Region in Sudden Unexplained Death and Sudden Unexpected Death in Epilepsy.

Sudden cardiac death (SCD) is a diagnostic challenge in forensic medicine. In a relatively large proportion of the SCDs, the deaths remain unexplained after autopsy. This challenge is likely caused by unknown disease mechanisms. Changes in DNA methylation have been associated with several heart diseases, but the role of DNA methylation in SCD is unknown. In this study, we investigated DNA methylation in two SCD subtypes, sudden unexplained death (SUD) and sudden unexpected death in epilepsy (SUDEP). We assessed DNA methylation of more than 850,000 positions in cardiac tissue from nine SUD and 14 SUDEP cases using the Illumina Infinium MethylationEPIC BeadChip. In total, six differently methylated regions (DMRs) between the SUD and SUDEP cases were identified. The DMRs were located in proximity to or overlapping genes encoding proteins that are a part of the glutathione S-transferase (GST) superfamily. Whole genome sequencing (WGS) showed that the DNA methylation alterations were not caused by genetic changes, while whole transcriptome sequencing (WTS) showed that DNA methylation was associated with expression levels of the GSTT1 gene. In conclusion, our results indicate that cardiac DNA methylation is similar in SUD and SUDEP, but with regional differential methylation in proximity to GST genes.

Gene expressions in cerebral palsy subjects reveal structural and functional changes in the gastrocnemius muscle that are closely associated with passive muscle stiffness.

Cerebral palsy (CP) is a non-progressive motor disorder that affects posture and gait due to contracture development. The purpose of this study is to analyze a possible relation between muscle stiffness and gene expression levels in muscle tissue of children with CP. Next-generation sequencing (NGS) of gene transcripts was carried out in muscle biopsies from gastrocnemius muscle (n = 13 children with CP and n = 13 typical developed (TD) children). Passive stiffness of the ankle plantarflexors was measured. Structural changes of the basement membranes and the sarcomere length were measured. Twelve pre-defined gene target sub-categories of muscle function, structure and metabolism showed significant differences between muscle tissue of CP and TD children. Passive stiffness was significantly correlated to gene expression levels of HSPG2 (p = 0.02; R2 = 0.67), PRELP (p = 0.002; R2 = 0.84), RYR3 (p = 0.04; R2 = 0.66), C COL5A3 (p = 0.0007; R2 = 0.88), ASPH (p = 0.002; R2 = 0.82) and COL4A6 (p = 0.03; R2 = 0.97). Morphological differences in the basement membrane were observed between children with CP and TD children. The sarcomere length was significantly increased in children with CP when compared with TD (p = 0.04). These findings show that gene targets in the categories: calcium handling, basement membrane and collagens, were significantly correlated to passive muscle stiffness. A Reactome pathway analysis showed that pathways involved in DNA repair, ECM proteoglycans and ion homeostasis were amongst the most upregulated pathways in CP, while pathways involved in collagen fibril crosslinking, collagen fibril assembly and collagen turnover were amongst the most downregulated pathways when compared with TD children. These results underline that contracture formation and motor impairment in CP is an interplay between multiple factors.

The stratum corneum transcriptome in atopic dermatitis can be assessed by tape stripping.

BACKGROUND: Skin biopsies represent a gold standard in skin immunology and pathology but can cause pain and induce scarring. Non-invasive techniques will facilitate study recruitment of e.g. patients with paediatric atopic dermatitis (AD), hand eczema or facial dermatitis. OBJECTIVE: By RNA sequencing, we examined whether the stratum corneum transcriptome in AD skin can be assessed by tape stripping, as compared to the epidermal transcriptome of AD in skin biopsies. To make the procedure clinically relevant tape strips were stored and shipped at room temperature for up to 3 days. METHODS: Nine adult Caucasian AD patients and three healthy volunteers were included. Tape samples were collected from non-lesional and lesional skin. Biopsies were collected from lesional skin and were split into epidermis and dermis. Total RNA was extracted, and shotgun sequencing was performed. RESULTS: Shotgun sequencing could be performed on skin cells obtained from two consecutive tape strips which had been stored and shipped at room temperature for up to three days. The most prominent differences between the tape strip and biopsy derived transcriptome were due to structural genes, while established molecular markers of AD, including CCL17, CCL22, IL17A and S100A7-S100A9, were also identified in tape strip samples. Furthermore, the tape strip derived transcriptome showed promise in also analysing the skin microbiome. CONCLUSION: Our study shows that the stratum corneum (SC) transcriptome of AD can be assessed by tape stripping the skin, supporting that this method may be central in future skin biomarker research. NCBI GEO data accession: GSE160501.

A comparative study of single nucleotide variant detection performance using three massively parallel sequencing methods.

Massively parallel sequencing (MPS) has revolutionised clinical genetics and research within human genetics by enabling the detection of variants in multiple genes in several samples at the same time. Today, multiple approaches for MPS of DNA are available, including targeted gene sequencing (TGS) panels, whole exome sequencing (WES), and whole genome sequencing (WGS). As MPS is becoming an integrated part of the work in genetic laboratories, it is important to investigate the variant detection performance of the various MPS methods. We compared the results of single nucleotide variant (SNV) detection of three MPS methods: WGS, WES, and HaloPlex target enrichment sequencing (HES) using matched DNA of 10 individuals. The detection performance was investigated in 100 genes associated with cardiomyopathies and channelopathies. The results showed that WGS overall performed better than those of WES and HES. WGS had a more uniform and widespread coverage of the investigated regions compared to WES and HES, which both had a right-skewed coverage distribution and difficulties in covering regions and genes with high GC-content. WGS and WES showed roughly the same high sensitivities for detection of SNVs, whereas HES showed a lower sensitivity due to a higher number of false negative results.

Whole genome and transcriptome sequencing of post-mortem cardiac tissues from sudden cardiac death victims identifies a gene regulatory variant in NEXN.

BACKGROUND: Sudden cardiac death (SCD) is a major public health problem and constitutes a diagnostic and preventive challenge in forensic pathology, especially for cases with structural normal hearts at autopsy, so-called sudden arrhythmic death syndrome (SADS). The identification of new genetic risk factors that predispose to SADS is important, because they may contribute to establish the diagnosis and increase the understanding of disease pathways underlying SADS. Pathogenic mutations in the protein coding regions of cardiac genes were found in relation to SADS. However, much remains unknown about variants in non-coding regions of the genome. METHODS AND RESULTS: In this study, we explored the potential of whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) to find DNA variants in SCD victims with structural normal hearts. With focus on the non-coding regulatory regions, we re-examined a cohort of 13 SADS and sudden unexplained death in infancy (SUDI) victims without disease causing DNA variants in recognized cardiac genes. The genetic re-examination of DNA was carried out using frozen tissue samples and WTS was carried out using five distinct formalin fixed and paraffin embedded (FFPE) cardiac tissue samples from each individual, including anterior and posterior walls of the left ventricle, ventricular papillary muscle, septum, and the right ventricle. We identified 23 candidate variants in regulatory sequences of cardiac genes, including a variant in the promotor region of NEXN, c.-194A>G, that was found to be statistically significantly (p < 0.05) associated with decreased expression of NEXN and cardiac hypertrophy. CONCLUSION: With the use of post-mortem FFPE tissues, we highlight the potential of using WTS investigations and compare gene expression levels with DNA variation in regulatory non-coding regions of the genome for a better understanding of the genetics of cardiac diseases leading to SCD.

Non-invasive prenatal paternity testing using a standard forensic genetic massively parallel sequencing assay for amplification of human identification SNPs.

Prenatal paternity testing often relies on invasive procedures that cause risk to both the mother and the foetus. Non-invasive, prenatal paternity testing by investigating paternally inherited single nucleotide polymorphisms (SNPs) in cell-free foetal DNA (cffDNA) in maternal plasma was performed at consecutive time points during early gestation. Plasma from 15 pregnant women was investigated at consecutive time points from gestational weeks (GWs) 4-20. The Precision ID Identity Panel and an Ion S5 Sequencer was used to analyse the cffDNA. Paternally inherited foetal SNP alleles were detected from GW7. The median foetal fractions were 0%, 3.9%, 5.1%, 5.2%, and 4.7% at GWs 4, 7, 12, 16, and 20, respectively. The corresponding median numbers of detected paternally inherited foetal autosomal SNP alleles were 0, 3, 9, 10, and 12, respectively. The typical (i.e. geometric mean) paternity indices at GW12 and GW20 were 24 (range 0.0035-8389) and 199 (range 5.1-30,137), respectively. The method is very promising. However, the method can be improved by shortening the lengths of the PCR amplicons and increasing the number of SNPs. To our knowledge, this is the first study to successfully identify paternally inherited foetal SNP alleles at consecutive time points in early gestation independently of the foetal gender.

Sequencing Library Preparation from Degraded Samples for Non-illumina Sequencing Platforms.

Efficient methods for building genomic sequencing libraries from degraded DNA have been in place for Illumina sequencing platforms for some years now, but such methods are still lacking for other sequencing platforms. Here, we provide a protocol for building genomic libraries from degraded DNA (archival or ancient sample material) for sequencing on the Ion Torrent™ high-throughput sequencing platforms. In addition to a reduction in time and cost in comparison to commercial kits, this protocol removes purification steps prior to library amplification, an important consideration for work involving historical samples. Libraries prepared using this method are appropriate for either shotgun sequencing or enrichment-based downstream approaches.

Quantification of massively parallel sequencing libraries - a comparative study of eight methods.

Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out.

Comparison of manual and automated AmpliSeq™ workflows in the typing of a Somali population with the Precision ID Identity Panel.

The Precision ID Identity Panel was used to type 109 Somali individuals in order to obtain allele frequencies for the Somali population. These frequencies were used to establish a Somali HID-SNP database, which will be used for the biostatistic calculations in family and immigration cases. Genotypes obtained with the Precision ID Identity Panel were found to be almost in complete concordance with genotypes obtained with the SNPforID PCR-SBE-CE assay. In seven SNP loci, silent alleles were identified, of which most were previously described in the literature. The project also set out to compare different AmpliSeq™ workflows to investigate the possibility of using automated library building in forensic genetic case work. In order to do so, the SNP typing of the Somalis was performed using three different workflows: 1) manual library building and sequencing on the Ion PGM™, 2) automated library building using the Biomek®3000 and sequencing on the Ion PGM™, and 3) automated library building using the Ion Chef™ and sequencing on the Ion S5™. AmpliSeq™ workflows were compared based on coverage, locus balance, noise, and heterozygote balance. Overall, the Ion Chef™/Ion S5™ workflow was found to give the best results and required least hands-on time in the laboratory. However, the Ion Chef™/Ion S5™ workflow was also the most expensive. The number of libraries that may be constructed in one Ion Chef™ library building run was limited to eight, which is too little for high throughput workflows. The Biomek®3000/Ion PGM™ workflow was found to perform similarly to the manual/Ion PGM™ workflow. This argues for the use of automated library building in forensic genetic case work. Automated library building decreases the workload of the laboratory staff, decreases the risk of pipetting errors, and simplifies the daily workflow in forensic genetic laboratories.

ISO 17025 validation of a next-generation sequencing assay for relationship testing.

The HID-Ion AmpliSeq™ Identity Panel is a next-generation sequencing assay with 90 autosomal and 34 Y-chromosome SNPs that are amplified in one PCR step and subsequently sequenced using the Ion Personal Genome Machine (Ion PGM™) System. This assay was validated for relationship testing in our ISO 17025 accredited laboratory in 2015. Here, the essential parts of the validation report submitted to the Danish Accreditation Fund are presented. A total of 100 unrelated Danes were typed in duplicates and the locus balance, heterozygote balance (Hb) and noise levels were analysed in detail. Two loci were disregarded for casework because genotyping was uncertain. Hb for rs7520386 was skewed and high levels of noise were observed in rs576261. Three general acceptance criteria for analysis of single-source samples were defined: (i) sequencing depth > 200 reads, (ii) noise level < 3% and (iii) Hb > 0.3. A Python script named SNPonPGM was developed to assist the analyst by highlighting loci that do not fulfil the general acceptance criteria. Furthermore, SNPonPGM has functions that reduce the hands-on time of the reporting officer to a few minutes per case. Mixtures with DNA from two individuals in a 1:24 ratio were readily identified using the three criteria and the SNPonPGM script.