Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method

Abstract

Sexual assault evidence often contains sperm cells, which are typically separated from nonsperm cells using manual differential lysis procedures. The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment. DNA yields were determined by qPCR, followed by multiplex STR amplification, CE analysis, and mixture interpretation. The automated method was capable of effective cell separation, producing DNA yields sufficient for STR amplification. Further, sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures, with 6-8× fewer female alleles detected (median 1.5 alleles). Ultimately, using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.

Keywords: Forensic sciences; QIAcube; automated DNA extraction; differential lysis; forensic genetics; mixture analysis; sexual assault evidence; sperm lysis.

Figure 1.

Total DNA yields from three differential cell lysis/DNA extraction methods. (A) The automated QIAcube method produced higher nonsperm fraction average DNA yields than the manual QIAGEN method, but this was not statistically significant (P = 0.09). The traditional manual organic method produced higher yields than both the automated QIAcube (*P = 0.02) and the manual QIAGEN (**P = 5.8 × 10⁻⁵) methods. (B) None of the three methods tested produced sperm fraction DNA yields that were statistically different when compared to each other (P = 0.29). n = 11 for automated QIAcube and manual QIAGEN, n = 10 for manual organic.

Figure 2.

Sperm fraction total human:male DNA ratios from three differential cell lysis/DNA extraction methods. The manual QIAGEN method consistently resulted in higher human:male DNA ratios, indicating a more substantial female contribution remaining in the sperm fraction for this method and better separation for samples processed using the automated QIAcube and manual organic methods. n = 11 for automated QIAcube, n = 10 for manual QIAGEN and manual organic.

Figure 3.

Representative sperm fraction electropherograms (blue channel) from three differential cell lysis/DNA extraction methods. (A) The automated QIAcube method is the only differential cell lysis/extraction method that resulted in a single-source STR profile for the sample shown. An increase in the number of female donor alleles was observed when the same mixture sample was processed using the manual QIAGEN method (B) or the manual organic method (C). STR alleles attributed to the female donor are circled.