Assigning forensic body fluids to donors in mixed body fluids by targeted RNA/DNA deep sequencing of coding region SNPs.

Biological traces found at crime scenes are analysed not only to genetically identify the donor(s) but also to determine the composition of the stain. For some cases, it is essential to associate a body fluid with a donor. Especially in mixed body fluid stains, but also in body fluid stains that appear to be single-source, this may be of importance. Linking a DNA profile (sub-source level) with evidence from a presumptive test or mRNA analysis (source level) is not straightforward. Our results support that associating donors and body fluids by means of comparing mixture ratios in RNA and DNA is not recommended. We introduce a set of 35 coding region SNPs (cSNPs) in body fluid-specific mRNA transcripts that represent a direct link between the body fluids and their donors. The discrimination power of the cSNPs was estimated based on allele frequencies calculated from a population sample (n = 188), and we investigated the practical application of the cSNPs in different scenarios. The results demonstrate that more cSNPs are needed to improve the discrimination power. However, the findings are promising as we were able to associate donors with body fluids in mixtures of different body fluids as well as in stains where both donors have contributed the same body fluid, e.g. a blood-blood mixture. In addition, the cSNP assay can be used for body fluid identification. The results of this proof-of-concept study support the use of cSNPs to assign body fluids to the respective donors.

The relationship between fatty acid profiles in milk identified by Fourier transform infrared spectroscopy and onset of luteal activity in Norwegian dairy cattle.

To investigate the feasibility of milk fatty acids as predictors of onset of luteal activity (OLA), 87 lactations taken from 73 healthy Norwegian Red cattle were surveyed over 2 winter housing seasons. The feasibility of using frozen milk samples for dry-film Fourier transform infrared (FTIR) determination of milk samples was also tested. Morning milk samples were collected thrice weekly (Monday, Wednesday, Friday) for the first 10 wk in milk (WIM). These samples had bronopol (2-bromo-2-nitropropane-1,3-diol) added to them before being frozen at -20°C, thawed, and analyzed by ELISA to determine progesterone concentration and the concentrations of the milk fatty acids C4:0, C14:0, C16:0, C18:0, and cis-9 C18:1 as a proportion of total milk fatty acid content using dry-film FTIR, and averaged by WIM. Onset of luteal activity was defined as the first day that milk progesterone concentrations were >3 ng/mL for 2 successive measurements; the study population was categorized as early (n=47) or late (n=40) OLA, using the median value of 21 DIM as the cutoff. Further milk samples were collected 6 times weekly, from morning and afternoon milkings, these were pooled by WIM, and one proportional sample was analyzed fresh for fat, protein, and lactose content by the dairy company Tine SA, using traditional FTIR spectrography in the wet phase of milk. Daily energy-balance calculations were performed in 42 lactations and averaged by WIM. Animals experiencing late OLA had a more negative energy balance in WIM 1, 3, 4, and 5, with the greatest differences been seen in WIM 3 and 4. A higher proportion of the fatty acids were medium chained, C14:0 and C16:0, in the early than in the late OLA group from WIM 1. In WIM 4, the proportion of total fatty acid content that was C16:0 predicted late OLA, with 74% sensitivity and 80% specificity. The long-chain proportion of the fatty acids C18:0 and cis-9 C18:1 were lower in the early than in the late OLA group. Differences were greatest in WIM 4 and 5. Differences in concentrations of cis-9 C18:1 were seen between the groups from WIM 1. No relationship was seen between OLA and milk concentrations of either protein or fat, or between OLA and the milk fat:protein ratio. The differences in milk fatty acid proportions between the 2 groups are most likely related to differences in energy balance. The study shows that frozen milk samples can be tested for fatty acids by FTIR spectroscopy and that FTIR spectroscopy of milk can be used to provide real-time information about cow reproductive function.

Is it possible to predict the origin of epithelial cells? - A comparison of secondary transfer of skin epithelial cells versus vaginal mucous membrane cells by direct contact.

In alleged sexual assault and rape cases, the focus has often been to collect samples from the victim's body, for detection of body fluids or skin cells from the offender. But in many cases intimate body samples from the perpetrator(s) can also be informative. However, in cases where the female victim claims vaginal penetration, the defendant may display an alternative explanation to the DNA findings, i.e. that the victim's skin cells has been secondarily transferred to his penis. We hypothesized that female DNA will be detected in a significantly greater amount on swabs from penis after intercourse than after secondary transfer by skin contact. Fourteen male-female couples were recruited to test the above hypothesis, by collecting penile swabs from 3 specified anatomical locations: Glans, shaft, and the coronal sulcus, after two different situations: Vaginal intercourse and secondary transfer of epithelial cells by skin contact. The results show that penile swabs following intercourse produce significantly higher DNA concentrations than after secondary transfer by skin contact. Our results, indicates which of the anatomical regions is best suited for sampling. The DNA profiling results show a preponderance of female profiles over male profiles following intercourse compared to secondary skin contact. Based on these data, it is possible to make a statistical model to distinguish between samples taken after intercourse and samples taken after secondary transfer by skin contact based on the amount of female DNA and mixture proportion (Mx) between female and male DNA in samples collected from penis swabs.

Body fluid identification and assignment to donors using a targeted mRNA massively parallel sequencing approach - results of a second EUROFORGEN / EDNAP collaborative exercise.

In a previous EUROFORGEN/EDNAP collaborative exercise, we tested two assays for targeted mRNA massively parallel sequencing for the identification of body fluids/tissues, optimized for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms, respectively. The task of the second EUROFORGEN/EDNAP collaborative exercise was to analyze dried body fluid stains with two different multiplexes, the former Illumina 33plex mRNA panel for body fluid/tissue identification and a 35plex cSNP panel for assignment of body fluids/tissues to donors that was introduced in a proof-of-concept study recently. The coding region SNPs (cSNPs) are located within the body fluid specific mRNA transcripts and represent a direct link between the body fluid and the donor. We predicted the origin of the stains using a partial least squares discriminant analysis (PLS-DA) model, where most of the single source samples were correctly predicted. The mixed body fluid stains showed poorer results, however, at least one component was predicted correctly in most stains. The cSNP data demonstrated that coding region SNPs can give valuable information on linking body fluids/tissues with donors in mixed body fluid stains. However, due to the unfavorable performance of some cSNPs, the interpretation remains challenging. As a consequence, additional markers are needed to increase the discrimination power in each body fluid/tissue category.

Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.