Variation in assessments of suitability and number of contributors for DNA mixtures.

The interpretation of a DNA mixture (a sample that contains DNA from two or more people) depends on a laboratory/analyst's assessment of the suitability of the sample for comparison/analysis, and an assessment of the number of contributors (NoC) present in the sample. In this study, 134 participants from 67 forensic laboratories provided a total of 2272 assessments of 29 DNA mixtures (provided as electropherograms). The laboratories' responses were evaluated in terms of the variability of suitability assessments, and the accuracy and variability of NoC assessments. Policies and procedures related to suitability and NoC varied notably among labs. We observed notable variation in whether labs would assess a given mixture as suitable or not, predominantly due to differences in lab policies: if two labs following their standard operating procedures (SOPs) were given the same mixture, they agreed on whether the mixture was suitable for comparison 66% of the time. Differences in suitability assessments have a direct effect on variability in interpretations among labs, since mixtures assessed as not suitable would not result in reported interpretations. For labs following their SOPs, 79% of assessments of NoC were correct. When two different labs provided NoC responses, 63% of the time both labs were correct, and 7% of the time both labs were incorrect. Incorrect NoC assessments have been shown to affect statistical analyses in some cases, but do not necessarily imply inaccurate interpretations or conclusions. Most incorrect NoC estimates were overestimates, which previous research has shown have less of an effect on likelihood ratios than underestimates.

DNAmix 2021: Laboratory policies, procedures, and casework scenarios summary and dataset.

DNAmix 2021 is a large-scale study conducted to evaluate the extent of consistency and variation among forensic laboratories in the interpretation of DNA mixtures, and to assess the effects of various potential sources of variability. This study utilized a multi-phasic approach designed to collect information about participating laboratories, laboratory policies, and their standard operating procedures (SOPs). It also characterizes the degree of variation in assessments of suitability and number of contributors as well as in comparisons and statistical analyses of DNA mixture profiles. This paper specifically details the study design and the data collected in the first two phases of the study: the Policies & Procedures (P&P) Questionnaire and the Casework Scenarios Questionnaire (CSQ). We report on the variation in policies and SOPs for 86 forensic laboratories-including information about their DNA workflows, systems, and type of statistics reported. We also provide details regarding various case-scenario specific decisions and the nature of mixture casework for 83 forensic laboratories. The data discussed in this article provide insight into the state of the field for forensic DNA mixture interpretation policies and SOPs at the time of the study (2021-2022).

Effect of Sterilants on Amplification and Detection of Target DNA from Bacillus cereus Spores.

To conceal criminal activity of a bioterrorist or agroterrorist, the site of pathogen generation is often treated with sterilants to kill the organisms and remove evidence. As dead organisms cannot be analyzed by culture, this study examined whether DNA from sterilant-treated Bacillus cereus spores was viable for amplification. The spores were exposed to five common sterilants: bleach, Sterilox®, oxidizer foam (L-Gel), a peroxyacid (Actril®), and formaldehyde vapor. The spores were inoculated on typical surfaces found in offices and laboratories to test for environmental effects. It was found that the surface influenced the efficiency of recovery of the organisms. The DNA isolated from the recovered spores was successfully detected using RT-qPCR for all treatments except for formaldehyde, by amplifying the phosphatidylinositol phospholipase C and sphingomyelinase genes. The results demonstrated that evidence from sites treated with sterilants can still provide information on the uncultured organism, using DNA amplification.

MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment.

The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5'-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification. The amplification conditions were modified by replacing AmpliTaq Gold with TaKaRa Ex Taq® HS, along with an enhanced PCR buffer system. The resulting method was compared to the recommended protocol and to a conventional non-MPS approach used in an operating forensic DNA laboratory. The modified amplification conditions gave equivalent or improved results, including when amplifying low amounts of DNA template from hair shafts which are a routine evidence type in forensic mtDNA cases. Amplification products were successfully sequenced using an MPS approach, addressing sensitivity of library preparation, evaluation of precision and accuracy through repeatability and reproducibility, and mixture studies. These findings provide forensic laboratories with a robust and improved enrichment method as they begin to implement the D-loop protocol from Illumina. Given that Ex Taq® HS is a proofreading enzyme, using this approach should allow for improved analysis of low-level mtDNA heteroplasmy.