Study on efficiency of the half-volume GlobalFiler™ PCR protocol: Internal validation, degraded trace analysis, and comparison with full-volume protocol using casework samples.

Due to its versatility, the GlobalFiler™ PCR Amplification Kit is commonly used in forensic analyses. The half-volume protocol used in our laboratory was validated by manually and automatically assembled PCRs involving increased number of PCR cycles. Validation parameters included tests of sensitivity, stability, reproducibility, repeatability, and casework study, in accordance with the guidelines of the Scientific Working Group on DNA Analysis Methods and the European Network of Forensic Science Institutes. To examine the efficiency of our custom half-volume and the manufacturer's full-volume protocol, we also compared the results of 266 casework samples amplified two times with both protocols. Our results demonstrated the high discriminatory power and robustness of the kit, as all the studied parameters provided appropriate results when the optimal 0.5 ng DNA input was used, regardless of the PCR preparation method. In the case of undegraded samples, DNA inputs in a range of 1-0.075 ng produced DNA profiles in an optimal RFU range without significant allelic dropout for both PCR preparation methods. Regarding the tests on degraded casework samples, the average locus dropouts for lowly, moderately, and highly degraded samples were 12.05%, 19.2%, and 33.79%, respectively, proving again the kit's abilities even in half-volume reaction mix. This protocol caused no loss of information, as comparing its results to the full-volume protocol's, the recovery of alleles showed no statistical difference. Based on our results, the GlobalFiler™ kit's numerous positive properties proved valid in the case of our half-volume protocol as well.