Whole-genome sequencing of artificial single-nucleotide variants induced by DNA degradation in biological crime scene traces.

The aim of this study was to identify artificial single-nucleotide variants (SNVs) in degraded trace DNA samples. In a preliminary study, blood samples were stored for up to 120 days and whole-genome sequencing was performed using the Snakemake workflow dna-seq-gatk-variant-calling to identify positions that vary between the time point 0 sample and the aged samples. In a follow-up study on blood and saliva samples stored under humid and dry conditions, potential marker candidates for the estimation of the age of a blood stain (= time since deposition) were identified. Both studies show that a general decrease in the mean fragment size of the libraries over time was observed, presumably due to the formation of abasic sites during DNA degradation which are more susceptible to strand breaks by mechanical shearing of DNA. Unsurprisingly, an increase in the number of failed genotype calls (no coverage) was detected over time. Both studies indicated the presence of artificial SNVs with the majority of changes happening at guanine and cytosine positions. This confirms previous studies and can be explained by depurination through hydrolytic attacks which more likely deplete guanine while deamination leads to cytosine to thymine variants. Even complete genotype switches from homozygote 0/0 genotypes to the opposite 1/1 genotypes were observed. While positions with such drastic changes might provide suitable candidate markers for estimating short-term time since deposition (TsD), 11 markers were identified which show a slower gradual change of the relative abundance of the artificial variant in both blood and saliva samples, irrespective of storage conditions.

Detecting DNA damage in stored blood samples.

Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess DNA quality. From routine casework in forensic genetics, it was observed that DNA degradation in forensic samples such as blood samples stored under sub-optimal conditions leads to visible effects in multiplex analyses of short tandem repeat markers (STRs) due to decreased amplification efficiencies in longer amplicons. It was further noticed that degradation indices often remain below the value that is considered to be critical. Thus, the aim of this work was to systematically analyze this effect and to compare conventional qPCR assays with a modified qPCR approach using uracil DNA glycosylase (UNG) and DNA quality assessment methods based on electrophoresis. Blood samples were stored at three different storage temperatures for up to 316 days. Significantly increased DNA recovery was observed from samples stored at high temperatures (37 °C) compared samples stored at room temperature and 4 °C. We observed typical effects of degradation in STR analyses but no correlation between DI and storage time in any of the storage conditions. Adding UNG slightly increased the sensitivity of detecting DNA degradation in one of the qPCR kits used in this study. This observation was not confirmed when using a second qPCR system. Electrophoretic systems did also not reveal significant correlations between integrity values and time. Methods for detecting DNA degradation are usually limited to the detection of DNA fragmentation, and we conclude that degradation affecting forensic STR typing is more complex.

Secondary DNA transfer by working gloves.

With the development of highly sensitive STR profiling methods, combined with sound statistical tools, DNA analysis on the (sub-)source level is hardly ever seriously questioned in court. More often, the exact mode of DNA transfer to the crime scene is questioned. In burglary cases, in particular when gloves are worn, secondary DNA transfer is often discussed as explanation for finding a DNA profile matching the accused because it is well known that gloves can act as a potential vector for indirect DNA transfer. In this study we investigated the shedder status as a possible factor influencing the extent of secondary DNA transfer to a crime scene, with the person committing the crime wearing working gloves. Firstly, the shedder status for 40 participants (20 male, 20 female) was determined, following a previously published procedure. Good shedders (n = 12) were found to deposit a higher amount and quality of DNA onto objects, compared to bad shedders (n = 25). Secondly, participants were paired into four groups (good with good; good with bad; bad with good; bad with bad), each group consisting of five pairs. The first participant (P1) of each pair used working gloves to pack and carry a box to simulate a house move. Two days later, the second participant (P2) of the pair wore the same pair of gloves to simulate a burglary, using a screwdriver as a break-in tool. After taking swabs of the outside and inside of a glove (primary DNA transfer) and the handle of the screwdriver (secondary DNA transfer), full DNA analysis was performed. Our experiments show that good shedders, overall, deposit more DNA than bad shedders, both onto the outside and the inside of the glove, regardless of being P1 or P2. When conducting the experiments with two participants sharing the same shedder status, no significant differences occurred in the number of deposited alleles. In six out of 19 cases a DNA profile matching P1 was found (binary LR>106) on the screwdriver and in all six cases P1 was a good shedder. Our results indicate that the shedder status of an individual affects the extent of DNA transfer. They further confirm the possibility of an innocent person's DNA profile being found on an object they never handled.

A proof of principal study on the use of direct PCR of semen and spermatozoa and development of a differential isolation protocol for use in cases of alleged sexual assault.

Sexual assault samples are some of the most common samples encountered in forensic analysis. These samples can require a significant time investment due to differential extraction processes. We report on the first record of successful direct amplification of semen for STR analysis. Neat seminal fluid, dilutions ranging from 1:5 to 1:160 and GEDNAP samples were successfully amplified using a direct method. A mild differential isolation technique to enrich spermatozoa was developed and successfully implemented to separate and directly amplify a mixture of semen and female epithelial cells. Aliquots of samples subjected to the differential isolation protocol were stained with Haemotoxylin and Eosin for sperm scoring. Samples stained after PCR showed a complete lack of intact spermatozoa demonstrating that the cells are lysed during the PCR process. This paper demonstrates the potential to incorporate direct PCR in cases of sexual assault to more rapidly obtain results and achieve a higher sensitivity.