Analysis of low-input and degraded forensic DNA trace samples with NGS using the MiSeq FGx platform

This study addresses the challenge of profiling low-input and degraded DNA traces in forensic science, by evaluating the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx platform. The standard microsatellite-based genotyping technology used for profiling forensic traces is generally inadequate for dealing with these types of samples. On the other hand, next-generation sequencing (NGS) offers the possibility of sequencing short polymorphism-containing fragments that are consistent with degraded DNA. In this study, we optimized the PCR amplification and library normalization steps using the ForenSeq™ DNA Signature Prep Kit composed of STR and SNP markers contained in PCR fragments ranging from 63 to 481 bp long, with an average length of 140 bp, in order to increase the probability of detection in degraded DNA. We evaluated these optimizations using both references and casework samples. Our results indicate that degraded traces can be profiled by NGS using either a combination of STR and SNP markers or a SNP-only signature, with a sufficient number of validated markers to support reliable identification potential. This work provides valuable insights into the optimization of NGS protocols and supports the use of the ForenSeq™ DNA Signature Prep Kit in complex forensic investigations. However, the introduction of a hybrid or SNP-only signature will require scientific and legal adjustments before it can be used routinely, or it will remain reserved for very special cases.