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The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/ DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were

Regularly, STR results obtained with different PCR amplification kits are compared, for instance with old cases, when revisiting cold cases or when addressing cross-border crimes. It is known that differences in primer design for the same loci in different kits may give rise to null alleles or shifted alleles. In this study, the genotyping results of 2085 Dutch male samples were compared for six autosomal STR kits (Promega's PowerPlex(®) 16, ESX-16 and ESI-17 Systems, Qiagen's Investigator(®) ESSplex Kit and Applied Biosystems' AmpFlSTR(®) Identifiler and NGM PCR Amplification Kits). A total of 19 discordant autosomal genotyping results were obtained that were examined by sequence analysis using Roche-454 next generation sequencing and/or Sanger sequencing. A further 25 discordances were found and sequenced for the Amelogenin locus. The 24 samples showing the same primer binding site mutation at the Amelogenin locus were subjected to X-STR analysis in order to assess whether they could share a common origin, which appeared not to be the case. Based on the sequencing results, we set the final genotypes and determined the allele frequencies of 23 autosomal STRs for the Dutch reference database.

The autosomal short tandem repeat (STR) kits that are currently used in forensic science have a high discrimination power. However, this discrimination power is sometimes not sufficient for complex kinship analyses or decreases when alleles are missing due to degradation of the DNA. The Investigator HDplex kit contains nine STRs that are additional to the commonly used forensic markers, and we validated this kit to assist human identification. With the increasing number of markers it becomes inevitable that forensic and kinship analyses include two or more STRs present on the same chromosome. To examine whether such markers can be regarded as independent, we evaluated the 30 STRs present in NGM, Identifiler and HDplex. Among these 30 markers, 17 syntenic STR pairs can be formed. Allelic association between these pairs was examined using 335 Dutch reference samples and no linkage disequilibrium was detected, which makes it possible to use the product rule for profile probability calculations in unrelated individuals. Linkage between syntenic STRs was studied by determining the recombination fraction between them in five three-generation CEPH families. The recombination fractions were compared to the physical and genetic distances between the markers. For most types of pedigrees, the kinship analyses can be performed using the product rule, and for those cases that require an alternative calculation method (Gill et al., Forensic Sci Int Genet 6:477-486, 2011), the recombination fractions as determined in this study can be used. Finally, we calculated the (combined) match probabilities, for the supplementary genotyping results of HDplex, NGM and Identifiler.

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