DNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools have a tradeoff between false positives and false negatives and present a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools, including a consensus approach, METEORE (https://github.com/comprna/METEORE), based on the combination of the predictions from two or more tools that shows improved accuracy over individual tools. Snakemake pipelines are also provided for reproducibility and to enable the systematic application of our analyses to other datasets.

Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.

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DNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools showed a tradeoff between false positives and false negatives, and presented a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools and proposed a new method, METEORE (https://github.com/comprna/METEORE), based on the combination of the predictions from two or more tools that has improved accuracy over individual tools. Snakemake pipelines are provided for reproducibility and to enable the systematic application of our analyses to other datasets.

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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.

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