Exploring the relationship of higher order DNA mixtures using autosomal STR and Y chromosome STR multiplexes

Abstract

Forensic laboratories like the Institute of Environmental Science and Research Ltd (ESR) are frequently tasked with sample analysis containing very low quantities of deoxyribonucleic acid (DNA), and an increasing number of different contributors. This can range from simple two-person DNA mixtures and up to three or more which are commonly known as higher-order DNA mixtures. Increasing the number of contributors or including biologically related contributors within a mixture, also increases the complexity of their DNA profile being interpreted. This is a recurring issue when investigating criminal cases like sexual assault which have devastating effects on the victim’s physical and mental well-being. This research thesis aims to investigate the relationship of higher order DNA mixtures using both autosomal short tandem repeats (STR) and Y-STR multiplexes. It was achieved following standard ESR casework protocols whilst simultaneously taking advantage of the more discriminating AmpFℓSTR™ IdentifilerTM Plus (ID+) and Yfiler™ Plus (YF+) PCR Amplification Kit from ThermoFisher. The results show that the major male identified on the Y chromosome specific DNA profile corresponds to the same major male contributor found on the autosomal DNA profile. This was found to occur within higher order mixtures of non-related contributors, in the absence or presence of excess female DNA and also higher order mixtures of biologically related contributors. A key result observed came from the three mixture sets including the presence of excess female DNA. These three mixture sets rotated through the same male contributors, but were amplified with three different female contributors. This further reinforces our hypothesis as the presence of three non-related females in excess had no impact at all on the capability to identify the major male contributor using YF+. This relationship could be observed at optimal template amounts for both multiplexes, but also at 0.5 ng for ID+ and 0.1 ng for YF+. It was easier to observe this relationship when the mixture ratio between the major and minor contributors was higher around 10: 1 or 12.5: 1 compared to if it was 5: 1: 1. Below these two template amounts for both multiplexes a great range of effects began to cause the resulting DNA profiles to gradually decrease their capability until they become uninterpretable. These effects include sharing of alleles, minor alleles falling in stutter positions, drop-out, and decreased ratio conservation as the template amount was lowered, but also the difference in obtaining results from higher molecular weighted loci compared to lower molecular weighted loci. The effects of sharing are compounded for the biologically related mixture set including a father and two sons causing these DNA profiles to be harder to interpret. These results demonstrate how specific higher-order mixture solutions behave; aiding forensic scientists in better informing the interpretation of results obtained from commonly encountered forensic casework samples. This in turn will allow more evidential inference to be drawn from these types of mixtures aiding in criminal investigations and the judiciary system.