An investigation of downstream processing methods for challenging skeletal samples.

While skeletal remains are known for their resilience and often serve as the final source of information for unidentified human remains (UHRs), the traditional downstream processing of these samples is challenging due to their low template nature, DNA degradation, and the presence of PCR inhibitors, typically resulting in limited probative information. To address this issue, advanced genotyping methods can be explored to retrieve additional genetic information from these challenging samples to maximize investigative leads. Therefore, this study investigated the effectiveness of three advanced genotyping methods and assessed their suitability with compromised skeletal samples: 1) targeted next generation sequencing (NGS) of both STRs and SNPs using the ForenSeq® DNA Signature Prep chemistry, 2) targeted NGS of SNPs using the ForenSeq® Kintelligence kit, and 3) SNP genotyping using a microarray via the Infinium Global Screening Array. The genotype recovery and added investigative leads were compared across all methods. All three approaches demonstrated success with the challenging skeletal samples used in this study. Specifically, the ForenSeq® DNA Signature Prep chemistry outperformed traditional STR typing by improving the recovery of CODIS core loci. Additionally, the ForenSeq® Kintelligence kit and Infinium Global Screening Array provided eligible results for forensic investigative genetic genealogy (FIGG) searching. Based on these successes, we have developed a proposed workflow for downstream processing of challenging skeletal samples. Following the guidelines of the US Department of Justice, the recovery of the CODIS core loci should be attempted through traditional CE-based methods or a NDIS-approved NGS chemistry, such as ForenSeq® DNA Signature Prep. Alternatively, a mitochondrial DNA profile may be uploaded to CODIS for comparisons in UHR cases. However, if no probative information is developed from the forensic profile uploaded to CODIS, then FIGG methods can be implemented using the Infinium Global Screening Array for high-quality skeletal samples (DNA concentrations ≥ 0.5 ng/µL) or the ForenSeq® Kintelligence chemistry for low-template skeletal remains (DNA concentration ≤ 0.5 ng/µL). These findings provide valuable insight into the suitability and efficacy of advanced genotyping methods, offering promising opportunities for enhancing the investigation of cases involving UHRs.