Analyzing degraded DNA and challenging samples using the ForenSeq™ DNA Signature Prep kit.

Typing short tandem repeats (STRs) is the basis for human identification in current forensic testing. The standard method uses capillary electrophoresis (CE) to separate amplicons by length and fluorescent labeling. In recent years new methods, including massively parallel sequencing (MPS), have been developed which increased the discriminative power of STRs through sequencing. MPS also offers the opportunity to test more genetic markers in a run than is possible with standard CE technology. Verogen's ForenSeq™ DNA Signature Prep kit includes over 150 genetic markers [STRs and single nucleotide polymorphisms (SNPs)]. Further, MPS separation depends on sequences rather than lengths; therefore, amplicons can be small or even of the same lengths. These improvements are advantageous when testing challenging forensic samples that could be severely degraded. This study tested the ForenSeq™ DNA Signature Prep kit in repeated experimental runs on series of degraded DNA samples, ranging from mild to severe degradation, as well as 24 mock case-type samples, derived from bones, blood cards, and teeth. Despite passing the quality metrics, positive controls (2800 M) showed drop-outs at some loci, mostly SNPs. Sequencing DNA samples repeatedly in two experimental runs as well as sequencing one pooled library in triplicate led to the assumption that spurious alleles of the Y-STRs in this study were not a result of sequencing artifacts but could be due to sequence structures (e.g. duplications, palindromes) of the Y-chromosome and/or might be accumulated during library preparation. Two sets of serially degraded DNA samples revealed that dropped-out loci were primarily loci with long amplicons as well as low read numbers (coverage), e.g. PentaE, DXS8378, and rs1736442. STRs started to drop out at degradation indices (DIs) > 4. However, severely degraded DNA (DI: 44) still resulted in 90% of the 20 CODIS loci, while only 35% were obtained using Promega's PowerPlex® Fusion kit, a current standard CE kit. Mock case-type samples confirmed these results. ForenSeq™ DNA Signature Prep kit demonstrated that it can be successfully used on degraded DNA samples. This study may be helpful for other laboratories assessing and validating MPS technologies.