Investigating SNP typing using alternative reference materials with the FORCE panel and QIAseq® chemistry

Advancements in forensic science have introduced single nucleotide polymorphism (SNP) markers as crucial tools in kinship, ancestry, and identity testing, and in predicting phenotypic traits. The emergence of Forensic Genetic Genealogy (FGG) and massively parallel sequencing (MPS) technologies have further enhanced the utility of SNP markers, generating interest in their application within the forensic community. The FORensic Capture Enrichment (FORCE) panel, targeting 5497 SNPs including those associated with ancestry, phenotype, identity, and kinship, is specifically designed for direct kinship comparisons and the recovery of autosomal markers for direct identification in cases where traditional short tandem repeat (STR) typing is unsuitable. This study aimed to evaluate alternative reference materials-hair roots, hair shafts, and fingernail clippings-using the FORCE panel and QIAseq® chemistry for direct identification. We assessed SNP recovery and concordance between these materials and buccal swabs, and we compared predictions for phenotype, Y-haplogroup, and biogeographic ancestry. Additionally, the study examined the concordance and performance on two sequencing platforms: MiSeq FGx and NextSeq 550. Our results demonstrated high (99.62-100 %) SNP concordance rates between alternative reference materials and buccal swabs, with fingernail samples showing the highest SNP recovery and concordance of the alternate reference materials. Phenotypic, ancestry, and Y-haplogroup ancestry predictions from alternative materials were 100 % consistent with those from buccal samples. However, some discrepancies in phenotype predictions were noted when comparing predictions to self-reported data. Both sequencing platforms provided comparable results, with NextSeq 550 mid-output kit offering 3.5X higher coverage and potential cost-saving advantages through greater sample multiplexing. In conclusion, the FORCE panel, combined with QIAseq® chemistry, effectively profiles SNPs from various alternative reference materials, offering reliable concordance and comprehensive genotype recovery even from low-input and degraded samples. This highlights its potential application in forensic investigations where traditional reference samples are unavailable.