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Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836–0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father–son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

An alternative approach to conventional protein-based body fluid identification is gene expression profiling analysis. In the present work, we report the development of sensitive and robust multiplex quantitative reverse transcriptase-PCR assays for the identification of blood, saliva, semen, and menstrual blood. Each body fluid assay comprises a triplex system that detects transcripts from two body fluid-specific genes and a housekeeping gene GAPDH. The body fluid-specific genes include erythroid d-aminolevulinate synthase (ALAS2) and b-spectrin (SPTB) for blood, statherin (STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1) and protamine 2 (PRM2) for semen, and matrix metalloproteinase 7 (MMP7) and matrix metalloproteinase 10 (MMP10) for menstrual blood. Normalization of both body fluid-specific genes to the housekeeping gene by means of appropriate cycle threshold metrics ensures the high specificity of each assay for its cognate body fluid.

RNA expression patterns, including the presence and relative abundance of particular mRNA species, provide cell and tissue specific information that could be used for body fluid identification. In this report, we address perceived concerns on the stability, and hence recoverability, of RNA in forensic samples. Stains were prepared from blood, saliva, semen, and vaginal secretions and exposed to a range of environmental conditions from 1 to 547 days. The persistence and stability of RNA within each type of body fluid stain were determined by quantitation of total RNA, and reverse transcriptase-polymerase chain reaction (RT-PCR) using eight different mRNA transcripts from selected housekeeping and tissue-specific genes. The results demonstrate that RNA can be recovered from biological stains in sufficient quantity and quality for mRNA analysis. On average, several hundred nanograms of total RNA was recovered from 50-microL-sized blood and saliva stains, 1 microg from a 50-microL semen stain and nearly 70 microg from a whole vaginal swab. Messenger RNA is detectable in some samples stored at room temperature for at least 547 days. The environmental samples that were protected from direct rain impact exhibited housekeeping and tissue specific mRNA recoverability up to 7 days (saliva and semen), 30 days (blood), or 180 days (vaginal swab). Additionally, rain had a detrimental effect on the recoverability of blood (3 days), saliva (1 day), semen (7 days), and vaginal secretions (3 days) specific transcripts, with one of the mRNA species (the semen marker PRM2) not being detectable after 1 day.

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The ability to determine the time since deposition of a bloodstain found at a crime scene could prove invaluable to law enforcement investigators, defining the time frame in which the individual depositing the evidence was present. Although various methods of accomplishing this have been proposed, none has gained widespread use due to poor time resolution and weak age correlation. We have developed a method for the estimation of the time since deposition (TSD) of dried bloodstains using UV-VIS spectrophotometric analysis of hemoglobin (Hb) that is based upon its characteristic oxidation chemistry. A detailed study of the Hb Soret band (λmax = 412 nm) in aged bloodstains revealed a blue shift (shift to shorter wavelength) as the age of the stain increases. The extent of this shift permits, for the first time, a distinction to be made between bloodstains that were deposited minutes, hours, days and weeks prior to recovery and analysis. The extent of the blue shift was found to be a function of ambient relative humidity and temperature. The method is extremely sensitive, requiring as little as a 1 µl dried bloodstain for analysis. We demonstrate that it might be possible to perform TSD measurements at the crime scene using a portable low-sample-volume spectrophotometer.