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.

D5S2500 is an ambiguously characterized STR: Identification and description of forensic microsatellites in the genomics age.

In the process of establishing short tandem repeat (STR) sequence variant nomenclature guidelines in anticipation of expanded forensic multiplexes for massively parallel sequencing (MPS), it was discovered that the STR D5S2500 has multiple positions and genomic characteristics reported. This ambiguity is because the marker named D5S2500 consists of two different microsatellites forming separate components in the capillary electrophoresis multiplexes of Qiagen's HDplex (Hilden, Germany) and AGCU ScienTech's non-CODIS STR 21plex (Wuxi, Jiangsu, China). This study outlines the genomic details used to identify each microsatellite and reveals the D5S2500 marker in HDplex has the correctly assigned STR name, while the D5S2500 marker in the AGCU 21plex, closely positioned a further 1643 nucleotides in the human reference sequence, is an unnamed microsatellite. The fact that the D5S2500 marker has existed as two distinct STR loci undetected for almost ten years, even with reported discordant genotypes for the standard control DNA, underlines the need for careful scrutiny of the genomic properties of forensic STRs, as they become adapted for sequence analysis with MPS systems. We make the recommendation that precise chromosome location data must be reported for any forensic marker under development but not in common use, so that the genomic characteristics of the locus are validated to the same level of accuracy as its allelic variation and forensic performance. To clearly differentiate each microsatellite, we propose the name D5S2800 be used to identify the Chromosome-5 STR in the AGCU 21plex.