In this study, PP35GY was evaluated and compared with PPF6C as a current 6-dye industry standard to determine if more genetic information could be obtained from degraded forensic samples and how the resulting match statistics would be affected. A sensitivity study was performed in addition to a degradation study using both artificially degraded and mock casework samples including bones, handled fired cartridge casings, and rootless hair shafts. The Quantifiler™ Trio degradation index (DI) was evaluated as an estimation of sample quality prior to amplification, where a DI less than one was not considered degraded and a DI greater than ten was considered severely degraded. All PP35GY samples were amplified in 25 µl reaction volumes at 29 cycles, injected with the default Spectrum CE 8C parameters, and analyzed in FaSTR™ DNA with the analytical thresholds defined in the PP35GY developmental validation [8]. With the exception of the sensitivity study, the optimal 1 ng template amount was targeted for PP35GY amplification when possible. All PPF6C analysis was completed according to internally validated methods and thresholds. Profile completeness based on the number of complete CODIS core STR loci and likelihood ratio strengths were compared. All statistical analyses were performed with the Caucasian allele frequencies reported in the NIST U.S. population data for 29 autosomal STR loci [10].

Seven-point dilution titrations of two reference samples, one female and one male, were used to evaluate the sensitivity of both kits. When template was less than 0.25 ng, the expanded 8-dye chemistry showed improvement in sensitivity as compared to PPF6C, indicated by higher profile completeness at lower input template amounts (Figure 2).

Twelve control DNA samples, from one reference male donor, were used to create artificially degraded DNA. Six were exposed to UV light with the SpectroLinker XL-1500 crosslinker at increasing amounts of time: 30, 60, 120, 240, 480, and 960 seconds; equivalent to 0.3, 0.7, 1.3, 2.6, 5.2, and 10.4 J/cm2, respectively. The remaining six samples were mechanically sheared with the Covaris M220 Focused-ultrasonicator in MicroTUBE-15 AFA Beads Screw Cap tubes at increasing treatment times of 42, 61, 80, 165, 250, and 335 seconds. All samples were amplified with the optimal 1.0 ng template DNA, except for the 960 second UV sample amplified with a maximum input of 0.78 ng. The DI increased with increasing treatment times for all samples while profile completeness decreased as expected. PP35GY outperformed PPF6C at the greatest degradation time points (Figure 3). These results demonstrate that the decreased PP35GY amplicon sizes allow for the generation of more complete profiles from degraded DNA.

Following the promising data from the artificially degraded samples, bones, handled fired cartridge casings, and rootless hair shafts were evaluated as typically challenging mock casework type samples (Figure 4).

1. Bones Four bones, two femurs and two occipital bones, were extracted from approximately 250 mg of bone powder with the proprietary Bode Bone Prep DNA extraction method. The two femur bones (Bones 1 and 2) generated the best profile results, while the two occipital bones (Bones 3 and 4) generated more varied profile results. PP35GY continued to outperform PPF6C when suboptimal template input (ng) and quality (DI) was amplified.

2. Fired Cartridge Casings Five handled fired cartridge casings (FCCs) were extracted with Bode X-traction for degraded DNA recovery. While the FCCs showed more variable results, the profile results of FCC3 and FCC4 both provide examples of the benefits of using the expanded dye-chemistry to gain more genetic information, and thus higher likelihood ratios, with input material of poorer quality.

3. Rootless Hair Shafts Two hair shaft samples were extracted with Bode X-traction for degraded DNA recovery with both samples able to be amplified with optimal template amount. For the sample with a low DI, both chemistries generated complete profiles with similar likelihood ratio strengths. However, as the DI increased, both profiles decreased in completeness and in likelihood ratio strength but with PP35GY outperforming PPF6C.

CONCLUSION

The results of this study support that with the smaller amplicon designs and increased multiplexing capacity, more genetic information and subsequently stronger match statistics can be obtained from input material of lower qualities and quantities. While the Y-STR markers were not discussed specifically in this article, PowerPlex® 35GY also enables scientists to obtain more genetic information while using less resources with the simultaneous amplification of 23 autosomal STRs and 11 Y-STRs in a single reaction. Overall, the expanded 8-dye chemistry presents notable improvements over current megaplex chemistries to better address the common limitations of typing degraded DNA.