2. Materials and methods2.1 Samples collection and extraction

The samples were extracted using the QIAamp® extraction DNA Mini protocol (Qiagen). The extraction procedure and Quantifiler® Trio DNA Quantification kit were followed in accordance with manufacturer’s recommendation. Ethical approval was granted by Ministry of Interior of Qatar and University of Central Lancashire “Ref STEM454″ for the PhD project.

2.2 PCR amplification & capillary electrophoresis

Two PCR amplification reactions were tested in this study following the PCR setup proportions recommended by the GlobalFiler™ PCR Amplification kit user guide. A full volume PCR of 25 μl was compared with a half volume PCR of 12.5 μl. Amplified samples were prepared for fragment analysis in capillary electrophoresis in capillary array ABI 3500 Genetic Analyzer.

2.3 Minimum threshold calculation

Data from 55 negative amplification controls were used to assess baseline noise and calculate a minimum detection and quantitation thresholds for the GlobalFiler™PCR kit at 29 cycles.

2.4 Half volume PCR reaction validation

In order to evaluate the PCR reaction of both GlobalFiler™, half volume experiment was conducted by amplifying 10 samples that had been profiled previously with a full volume reaction. The positive DNA controls of 2800 M, 9947A and the 10 Male DNA sample, were prepared and analysed parallel with serial dilutions reactions.

2.5 Sensitivity study using half volume PCR

Two DNA control samples, 9947A and positive control 007, were used in the sensitivity study. A serial dilution of the samples was prepared having DNA amounts of 1 ng, 750 pg, 500 pg, 250 pg, 125 pg, 62.5 pg, 31.25 and 15 pg. These were amplified, in triplicate.

2.6 Precision, reproducibility and accuracy studies

A total of 11 unrelated male samples including 2800, 9947A positive DNA control were used for the precision and accuracy study. Results from three different runs were analysed and compared to allelic ladder. Mean allele size and standard deviations were determined over three different runs and compared with the allelic ladder in order to illustrate the precision and accuracy of sample analysis.

2.7 Female/male, male/male DNA sample mixture

In the internal validation of GlobalFiler™ kit, a mixed male/female DNA controls (DNA 007 and 9947A and 2800) with known ratios were tested in triplicate. The mixture ratios included 1:1, 1:3, 3:1, 1:9, 9:1, 1:19 and 19:1, for 1 ng of mixed template DNA in triplicate.

2.8 Casework samples study

A total of 64 forensics DNA samples were used in this study. The casework samples from cases received in forensic Laboratory were used to assess the GlobalFiler™ kit. All samples were previously extracted using different extraction methods, and quantified using Quantifiler® Trio kit and profiled previously with Identifiler Plus™ kit.

2.9 Stutter analysis

Stutter percentages were calculated at each locus. This was previously analysed using 100 samples used for allele range determination experiment. Stutter percentages were calculated for each locus by dividing the stutter peak height by the parent peak height and multiplying it by 100.

2.10 Species specificity study

Non-human samples extracts were provided by UCLAN lab, which include dog, pig, horse, chicken, mouse, and rat.

5. Conclusion

These data demonstrate that the GlobalFiler™ PCR amplification kit generates high quality, reproducible, precise, accurate, and sensitive profiling STR data, even from sub-nanogram amounts of genomic DNA template from a variety of forensic samples including blood, semen and saliva stains, hair, muscle, tissues, bone and teeth. Analytical, stochastic and stutter threshold data were assessed for GlobalFiler™ in this study. The use of a half-reaction protocol would be operationally a robust method besides significant cost effectiveness.