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.

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.

Often bones are the only biological material left for the identification of human remains. As situations may occur where samples need to be stored for an extended period without access to cooling, appropriate storage of the bone samples is necessary for maintaining the integrity of DNA for profiling. To simulate DNA preservation under field conditions, pig rib bones were used to evaluate the effects of bone cleaning, buffer composition, storage temperature, and time on DNA recovery from bone samples. Bones were stored in three different buffers: TENT, solid sodium chloride, and ethanol-EDTA, at 20 °C and 35 °C for 10, 20, and 30 days. Bones were subsequently dried and ground to powder. DNA was extracted and quantified. Results show that temperature and storage time have no significant influence on DNA yield. DNA recovery from bones stored in solid sodium chloride or ethanol-EDTA was significantly higher compared to bones stored in TENT, and grinding of bones was facilitated by the extent of dehydration in solid sodium chloride and ethanol-EDTA compared to TENT. Overall, solid sodium chloride was found to be superior over ethanol-EDTA; when it comes to transportation, dry material such as salt eliminates the risk of leaking; it is non-toxic and in contrast to ethanol not classified as dangerous goods. Based on this study’s results, we recommend NaCl as a storage substrate for forensic samples in cases where no cooling/freezing conditions are available.