Expanding the forensic utility of the 94 ForenSeq identity SNPs through sequence-based microhaplotypes and an...

Next-generation sequencing (NGS) enables the detection of additional sequence variation within identity-informative single nucleotide polymorphism (iiSNP) amplicons beyond the targeted marker, allowing the characterisation of sequence-based haplotypes, including microhaplotypes (MHs), that may increase forensic informativeness. However, harmonised haplotype definitions, population frequency databases, and practical tools for routine implementation remain limited for the 94 iiSNPs included in the ForenSeq™ DNA Signature Prep Kit. In this study, sequence variation within the 94 ForenSeq iiSNP amplicons was characterised using NGS data from the Valencian Community dataset, representing the Eastern Iberian Population, together with phased high-coverage data from the 1000 Genomes Project. Resulting haplotypes were harmonised into common haplotype definitions across datasets, annotated, classified, and used to generate population frequency databases. Their forensic performance was evaluated and compared with conventional target SNP genotypes. Sequence-based analysis increased the number of detectable alleles from 188 theoretical SNP alleles to 268 sequence-based haplotypes in the Valencian Community dataset, with up to 390 haplotypes identified using the phased 1000 Genomes Project data. Depending on the population analysed, between 13 and 33 loci were classified as MHs. Incorporating variation within the flanking regions improved locus-specific forensic parameters, enhanced overall discriminatory power, and provided additional evidential value in kinship analyses and real forensic cases, specifically in human identification cases from the Spanish Democratic Memory Programme. Remarkably, these gains were achieved using sequence variation already captured by the existing assay, without requiring additional markers, laboratory procedures, or sequencing effort. In addition, we developed forenseqhaplo, an open-source and configurable workflow for automated haplotype extraction, filtering, assignment, and reporting directly from ForenSeq Universal Analysis Software v2.7 Flanking Region Reports. Altogether, the reference databases and analytical workflow presented here provide a practical framework for the routine implementation of sequence-based haplotypes, including MHs, in forensic identification and kinship analyses.