Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Papaver somniferum L. is cultivated for its edible seeds and for the production of alkaloids. A serious problem in seed trade and processing is the intentional mixing of excellent food-quality seeds with non-food-grade-quality seeds. Tracking the correct or illegitimate handling of seeds requires an efficient method for discrimination and individualization of poppy varieties. As in human and animal forensics, DNA variable regions containing short tandem repeats (STRs) located either in non-coding DNA or in gene sequences (EST-STRs) are preferred markers for discrimination between genotypes. Primers designed for 10 poppy EST-STR loci not analyzed so far were tested for their discriminatory ability on a set of 23 related P. somniferum L. genotypes. Thirty-three EST-STR alleles were identified together. Their polymorphic information content (PIC) values were in the range of 0.175-0.649. The PI value varied in the range of 0.140-0.669, and the cumulative PI was 1.2 × 10-5. PIsibs values varied between 0.436 and 0.820 and the cumulative value was lower (5.0 × 10-3). All analyzed genotypes were distinguished mutually, each with its own unique EST-STR profile. These newly developed EST-STR markers more effectively discriminated P. somniferum L. genotypes, even those genotypes whose DNA profiles were previously identical.

A survey to determine the presence of Mycobacterium spp. in the Abruzzo and Molise regions was conducted by testing samples from 124 badgers found dead or road-killed during the 2013-2021 period. Head lymph nodes were collected from all carcasses, as well as mediastinal lymph nodes from 20 of them, for bacteriological and molecular tests; tissues were inoculated onto a set of solid egg-based Lowenstein-Jensen media and in a liquid culture system (BACTEC) and were analyzed by polymerase chain reactions (PCRs). Organs and lymph nodes from 31 carcasses were collected for histological tests. During post-mortem examinations, macroscopic lesions consistent with a Mycobacterium tuberculosis complex (MTBC) and with nontuberculous mycobacteria (NTM) infections were not detected. Mycobacteria were isolated from four animals (3.22%). M. avium subsp. avium was isolated by head lymph nodes from two badgers (1.61%), M. avium subsp. paratuberculosis (0.80%) from one, and Mycobacterium spp. from another (0.80%). The significance of nontuberculous mycobacteria (NTM) in wildlife hosts in the absence of clinical signs and gross pathology has yet to be assessed. The most critical aspect came from isolates belonging to the Mycobacterium avium complex infection in wildlife due to the possible interference with tuberculin skin tests in cattle.

Forensic DNA typing has been widely accepted in the courts all over the world. This is because DNA profiling is a very powerful tool to identify individuals on the basis of their unique genetic makeup. DNA evidence is capable of not only identifying the presence of specific biospecimens in a crime scene, but it is also used to exonerate suspects who are innocent of a crime. Technological advancements in DNA profiling, including the development of validated kits and statistical methods have made this tool to be more precise in forensic investigations. Therefore, validated combined DNA index system (CODIS) short tandem repeats (STRs) kits which require very small amount of DNA, coupled with real-time polymerase chain reaction (PCR) and the statistical strengths are used routinely to identify human remains, establish paternity or to match suspected crime scene biospecimens. The road to modern DNA profiling has been long, and it has taken scientists decades of work and fine tuning to develop highly accurate testing and analyses that are used today. This review will discuss the various DNA polymorphisms and their utility in human identity testing.

BACKGROUND: Massively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses.
RESULTS: DNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened.
CONCLUSIONS: Although the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data.

The SeqStudio™ for human identification (HID) is a new benchtop capillary electrophoresis (CE) platform recently developed by Applied Biosystems for genotyping and sequencing short tandem repeat (STR) fragments. Compared to the previous series of CE systems developed by this maker, it is more compact and easier to use. Moreover, by allowing the detection of 4 to 8 fluorescent dyes, it seems to be fully compatible with the different kits of autosomal and gonosomal STR markers usually used in forensic genetics, which are available in trade and supplied by various manufacturers. However, being a new CE model, before its routine use in forensic genetics applications, it should undergo appropriate analytical validation studies in its own laboratories to understand its potential and limitations. A series of experiments on DNA samples coming from cell line controls, using the GlobalFiler™ IQC Amplification Kit, were carried out to meet this purpose. The SeqStudio™ Genetic Analyzer for HID\'s findings on genotyping reproducibility (precision and accuracy of sizing), sensitivity, signal variability between dyes (intra- and inter-color channel balance), and stutter ratios are reported. These findings confirm the validity of this new CE system and its capability to generate reliable results.

BACKGROUND: Short tandem repeats (STRs) have been used for various identity typing methods worldwide. They have high discrimination power in human identification in forensics, paternity testing, missed personal identification, genetic diseases, and gene regulatory functions. They have also been used to detect and monitor the stability of diseases, including various types of cancer. This study aimed to investigate the impact of leukemia on the detection and stability of STR markers.
METHODS: DNA was isolated from 30 participants (15 with chronic myeloid leukemia( CML) and 15 healthy controls) and used to amplify STR markers using specific primers.
RESULTS: We found that the blood of those with leukemia had more 9.3 and 9 alleles at the tyrosine hydroxylase 1 (TH01) marker than the blood of the healthy control samples. The results of this study will help researchers understand leukemia\'s effect on the detection and stability of STR markers in leukemic patients compared to healthy individuals.
CONCLUSIONS: Our results demonstrate that STR markers could become useful in genetic studies of leukemia cases.

The present research assessed how the physical and chemical changes associated with decomposition affect the detection and identification of blood and semen evidence, as well as subsequent DNA analysis. A feeder pig (postmortem interval (PMI) < 3 h) was placed within the Boston University Outdoor Research Facility for a period of 22 days. Human blood and semen were individually dispensed onto multiple areas of two cotton t-shirts; one layer of fabric was placed above and below the pig and a control. One of each sample type was collected per day for a period of 22 days from each location. It was observed that both sample types when collected from beneath the pig exhibited the greatest decline in enzymatic activity over the course of testing, followed by samples from beneath the control, which can be inferred from the increase in negative screening results compared to the other samples. Spermatozoa were observed in nearly all semen samples, even when all screening results were negative, which lead to the generation of comparable DNA profiles for nearly all semen samples typed. Genetic typing of the blood samples beneath the pig and control rarely yielded comparable data while the samples from above yielded full profiles for all but a few samples tested.

The identification of human remains is challenging mostly due to the bad condition of the remains and the available background information that is sometimes limited. The current case report is related to the identification of an unknown soldier from the Estonian War of Independence (1918-1920). The case includes an anthropological study of the remains, examinations of documents found with the exhumed remains, and kinship estimations based on archival documents, and DNA analyses. As the preliminary data pointed to remains of male origin, Y-chromosomal STR (short tandem repeat) analyses of 22 Y-STR loci were used to analyze the exhumed teeth. Reference samples from individuals from two paternal lineages were collected based on archival documents. Y-chromosomal STR results for the tooth samples were consistent with a patrilineal relationship to only one reference sample out of two proposed paternal lineages. Based on the provided pedigrees in the consistent case, the Y-STR results are approximately four million times more likely if the tooth sample originated from an individual related along the paternal line to the matching reference sample, than if the tooth sample originated from another person in the general population. Special considerations have to be met when limited evidence is available.

Rwanda is one of the smallest countries of Africa, where forensic genetic studies are rarely being conducted and very few DNA databases have been developed. Short tandem repeats (STRs) polymorphisms were investigated in 505 unrelated Rwandese by using the HUMDNA TYPING (Yanhuang) Kit. The following STRs were targeted: D3S1358, D13S317, D7S820, D16S539, SE33, D10S1248, D5S818, D21S11, TPOX, D1S1656, D6S1043, D19S433, D22S1045, D8S1179, Penta E, D2S441, D12S391, D2S1338, vWA, Penta D, TH01, D18S51, CSF1PO and FGA. The purpose of this study was to elucidate the genetic diversity and explore the potential of applying these 24 STR in 505 Rwandan population in forensics. A total of 360 alleles, with corresponding allele frequencies in the range from 0.001 to 0.442, were found in the Rwandan population. SE33 presented the highest polymorphism (PIC=0.921) among these 24 loci, whereas D13S317 presented the lowest one (PIC=0.671). No deviation from the Hardy-Weinberg equilibrium was observed for any of the 24 loci. The forensic parameters, including the combined power of discrimination (PD and the combined exclusion power, have demonstrated that this panel of 24 STRs is highly informative and useful for forensic applications such as individuals\' identification and paternity tests. Additionally, the genetic distances between Rwanda population and other 24 published populations were calculated based on 8 overlapping loci with the polygenetic tree revealing significant clusters in the populations associated with their geographic locations and their historical relationship.