Short tandem repeat (STR) based sequence typing of Entamoeba histolytica identifies STGA-D locus as a genetic marker, associated with disease outcomes.

Amoebiasis, caused by the enteric parasite Entamoeba histolytica has differential disease outcomes. The association of parasite genotypes with outcomes of amoebic infection is still a paradox and requires to be explored. The genetic information of infecting strains from endemic settings of different geographical regions is essential to evaluate the relation. Comparative genetics of E. histolytica clinical isolates from different disease outcomes have been explored based on two tRNA-linked STR loci (STGA-D and A-L). All of the repeat patterns in the A-L locus were newly identified and unique to Indian isolates. The majority of newly identified repeat patterns in STGA-D locus have outcome-specific distributions, predicting the emergence of disease-specific mutations in this target locus. Statistical analysis further reinforces this observation, as identified repeat patterns only from STGA-D but not A-L locus were significantly associated with disease outcomes. Phylogenetic analysis indicates independent segregation and divergence of tRNA-linked STR arrays for each STR locus.

Genetic Diversity of Kazakhstani Equus caballus (Linnaeus, 1758) Horse Breeds Inferred from Microsatellite Markers.

Understanding the genetic diversity and structure of domesticated horse (Equus caballus) populations is critical for long-term herd management and breeding programs. This study examines 435 horses from Kazakhstan, covering seven groups in three geographic areas using 11 STR markers. Identified are 136 alleles, with the mean number of alleles per locus ranging from 9 to 19. VHL20 is the most variable locus across groups, while loci HTG4, AHT4, AHT5, HTG7, and HMS3 are variable in most populations. The locus AHT5 in the Emba population shows the highest frequency of rare alleles, while the lowest frequency, 0.005, is observed in the Kulandy population. All loci were highly informative for the Kazakhstani populations of E. caballus, with PIC values higher than 0.5. Pairwise variations in Wright's FST distances show that the examined varieties have little genetic differentiation (0.05%), indicating a high degree of admixture and a continuing lineage sorting process. Phylogenetic and population structure analyses reveal three major clusters of Kazakh horses, representing (I) the Uralsk population of the Kushum breed and the monophyly of two groups: (II) the Kozhamberdy population of the Mugalzhar breed, and (III) the Mugalzhar-Kushum breed populations. Kazakhstani horse populations, while being regionally isolated, were recently in contact with each other.

Extraction of DNA from trace forensic samples with a modified lysis buffer and chitosan coated magnetic beads.

The trace amounts of human tissue cells or body fluids left at the crime scene are often mixed with inhibitors such as rust, pigments, and humic acid. The extraction of the DNA from the trace cells is crucial for the investigation of cases. Usually, specially designed magnetic nanoparticles were chosen by the case investigators to enrich and elute DNA, which was then used for polymerase chain reaction (PCR) and short tandem repeat (STR) analysis. The traditional approach often had the following problems, such as low DNA enrichment efficiency, possible DNA breakage, and complex operations. Here, the 1%(w/v) of chitosan (75% deacetylation degree) was used to modify the 50 nm magnetic nanoparticles to gain the Chitosan@Beads, which theoretically carried positively charged in the pH = 5 of lysis buffer so as to adsorb negatively charged DNA through electrostatic interactions. The XPS and FT-IR results demonstrated that chitosan was successfully attached to the surface of magnetic nanoparticles. A set of simulated samples, containing 20 mg/µL of humic acid, pigments, iron ions (Fe2+, Fe3+), and the coexistence of the above three substances, were prepared to simulate the case scene. Human bronchial epithelial cells were mixed with the 200 µL of the above simulated samples for DNA extraction. 400 µL of lysis buffer, 20 µL of proteinase K (10 mg/mL) and 20 µL of Chitosan@Beads solution (20 mg/mL) was used for cell disruption and DNA enrichment. The extraction sensitivity of Chitosan@Beads was confirmed to be 10 cells, superior to commercial reagent kits. The Chitosan@Beads@DNA can directly use for "In-situ PCR" with elution-free operations. The STR loci rate of DNA extracted by Chitosan@Beads was around 97.9%, higher than the commercial kit (66.7%). In short, we foresee here developed novel Chitosan@Beads and modified lysis buffer could provide a new model for the DNA extraction of forensic trace evidence.

Sequence-based allelic variations and frequencies for 22 autosomal STR loci in the Lebanese population.

This is the first study that characterizes the sequence-based allelic variations of 22 autosomal Short Tandem Repeat (aSTR) loci in a population dataset collected from Lebanon. Genomic DNA extracts from 195 unrelated Lebanese individuals were amplified with PowerSeq 46GY System Prototype. Targeted amplicons were subjected to DNA library preparation and sequenced on the Verogen MiSeq FGx Sequencing System. Raw FASTQ data files were processed by STRait Razor v3. Sequence strings were annotated according to the considerations of the DNA Commission of the International Society for Forensic Genetics (ISFG) and tabulated herein with their respective allelic frequencies and GeneBank accession and version numbers. The sequenced Lebanese dataset resulted in 429 distinct allelic sequences as compared to the 236 alleles identified by length only. The increase in the number of alleles was observed at 18 out of 22 aSTR loci and was attributed to the sequence variations residing in both the STR repeat motifs and flanking regions. The study uncovered 25 novel aSTR allelic sequences across 12 loci for which GenBank records did not previously exist in the STRSeq BioProject, PRJNA380127. For a concordance check, the length-based allelic calls derived from the full sequences were compared to those genotyped using capillary electrophoresis (CE) methods. Population genetic parameters relevant to the evaluation of forensic DNA evidence were assessed for the sequence-based data and compared to the parameters generated from the length-based information. Using the sequence-based data, Analysis of MOlecular VAriance (AMOVA), genetic distances, and population genetic structure were evaluated for 1231 individuals sampled from the Lebanese and four U.S. populations (African American, Asian, Caucasian, and Hispanic). The results were tabulated and visualized in a population tree, multidimensional scaling scatter plots, and bar plots. This newly established sequence-based database for the Lebanese population can be beneficial for extending NGS applicability to casework or paternity testing and assessing the strength of evidence for NGS-STR profiles. The described novel sequence variants at certain loci can further help in the effort to characterize the sequence diversity of STR markers from different populations around the world.

Analysis of short tandem repeat mutations in paternity cases from Masovian Voivodeship provinces form years 2018-2022 based on materials of the Department of Forensic Medicine, Medical University of Warsaw. / Analiza mutacji krótkich powtórzen tandemowych w sprawach spornego ojcostwa w woj. mazowieckim w latach 2018-2022 na podstawie materialów Katedry i Zakladu Medycyny Sadowej Warszawskiego Uniwersytetu Medycznego.

In paternity cases, genetic tests are of great importance as they allow to exclude or confirm paternity. As a result of paternity tests we can also obtain information on the frequency of short tandem repeat mutations, which are important in the statistical analysis of test results. A total of 468 cases of full paternity trios (mother, child and alleged father) were analysed from years 2018 - 2022 from the central part of Poland. For further analysis of the occurrence of the mutation 346 cases in which paternity was confirmed were qualified. DNA analysis was performed using the PowerPlex®Fusion 6C kit (Promega, USA). 36 mutations were observed in 13 of the 23 genetic markers analysed. 94.44% were one-step mutations and 5.56% were two-step mutations. Among those mutations, there were 18 insertions and 10 deletions, while in 8 cases it was not possible to determine whether an insertion or deletion occurred. There was also a significantly higher share of the father mutation in relation to the mother mutation at a ratio of 4.17:1.

Mutation rate evaluation at 21 autosomal STR loci: Paternity testing experience.

The short tandem repeats (STRs) or microsatellites are used for paternity testing and these sequences mutate more rapidlythanbulkDNAsequences. A total of 746 paternity cases were analysed to understand the mutation rate of 21 autosomal STR loci. We identified 41 mutations in 11 STR Loci with a maximum at SE33. No mutations occurred in the remaining 10 STR loci. The overall average mutation rate was estimated as 0.004523 and the estimated locus-specific mutation rate varied between 0.001214 and 0.016990. Among these 90.24% was accounted for single-step mutation, 2.44% for two steps, and 7.32 % for three or muti steps. The obtained data is crucial and could be helpful for ensuring the accuracy of DNA testing and interpretation.

The first data of allele frequencies for 23 autosomal STRs in the Ede ethnic group in Vietnam.

Settled in Vietnam, the Ede ethnic group exhibits unique heritages that belong to the Austronesian-speaking groups. In this study, we genotyped 23 autosomal short tandem repeat (STR) markers in 397 unrelated samples from the Ede population and recorded the first data of allele frequencies and forensic parameters for the Ede. Data analysis revealed high values of the combined discrimination power (0.999 999 999 999 999 999 999 999 950) and the exclusion power (0.999 999 999 999 998). Within the Ede population, D12S391 was indicated to be an informative autosomal STR marker for individual identification and paternity testing. A phylogenetic tree of the Ede and nine other Austronesian groups, spanning from Africa to Asia and Oceania, asserted a close relationship among these groups, especially among the Ede, Malaysian Malay, and Indonesian populations.

Validation and forensic application of a new 19 X-STR loci multiplex system.

The Microreader™ 19X Direct ID System was a newly developed multiplex PCR kit, which could detect 19 X-chromosomal STR loci (DXS6795, DXS9907, DXS6803, GATA172D05, DXS6807, GATA31E08, DXS7423, DXS6810, DXS101, DXS9902, DXS7133, DXS6800, DXS981, DXS10162, DXS6809, DXS10135, HPRTB, GATA165B12, DXS10079) and the sex determination locus of AMEL simultaneously. Different from other X-STR multiplex PCR kits, no linkage groups are included in this system, so the likelihood ratios could be calculated without the consideration of linkage groups. In this study, PCR conditions, sensitivity, species specificity, stability, DNA mixtures, concordance, stutter, sizing precision and population studies were conducted according to the SWGDAM developmental validation guidelines. The results indicated that this new X-STRs multiplex system was an efficient and reliable detection system, which could facilitate human kinship analysis and identification testing, as a powerful supplementary to autosomal STR kits.

Assessing false paternity risk in simulated motherless cases from more than 20 000 real exclusion trios.

BACKGROUND: When the mother's DNA profile is not available for paternity testing, there is a smaller probability that a locus will exclude an alleged father. This study aims to evaluate the risk of potential false paternity inclusions in motherless cases. STUDY DESIGN AND METHODS: More than 20 000 duos were generated by removing the maternal genotypes from exclusion trios. After recalculating paternity in these duos, any found inclusions would be false. RESULTS: The use of an appropriate number of loci, mutation model, and mutation rates to analyze motherless paternity cases was robust against false inclusions. A single potential false inclusion was observed in a case wherein kinship plays a role. This result highlights the importance of testing the mother when available and of obtaining information on family circumstances for the proper handling of cases involving related individuals. CONCLUSION: The guidelines we used here were sufficient to avoid false inclusions in a data set of more than 20 000 motherless cases.

Y-profile evidence: Close paternal relatives and mixtures.

We recently introduced a new approach to the evaluation of weight of evidence (WoE) for Y-chromosome profiles. Rather than attempting to calculate match probabilities, which is particularly problematic for modern Y-profiles with high mutation rates, we proposed using simulation to describe the distribution of the number of males in the population with a matching Y-profile, both the unconditional distribution and conditional on a database frequency of the profile. Here we further validate the new approach by showing that our results are robust to assumptions about the allelic ladder and the founder haplotypes, and we extend the approach in two important directions. Firstly, forensic databases are not the only source of background data relevant to the evaluation of Y-profile evidence: in many cases the Y-profiles of one or more relatives of the accused are also available. To date it has been unclear how to use this additional information, but in our simulation-based approach its effect is readily incorporated. We describe this approach and illustrate how the WoE that a man was the source of an observed Y-profile changes when the Y-profiles of some of his male-line relatives are also available. Secondly, we extend our new approach to mixtures of Y-profiles from two or more males. Surprisingly, our simulation-based approach reveals that observing a 2-male mixture that includes an alleged contributor's profile is almost as strong evidence as observing a matching single-contributor evidence sample, and even 3-male and 4-male mixtures are only slightly weaker.

SE33 locus as a reliable genetic marker for forensic DNA analysis systems

Background/aim: Genetic variation, an authentic tool of individual discrimination, is being used for forensic investigations worldwide. A missing result for even one out of 13-17 markers leads to an inconclusive report. Additional reliable markers are required to compensate such deficiencies. The SE33 locus has high genetic variability in different populations and is being used in forensic investigation systems in some countries. The purpose of the study was to assess the viability of use of the SE33 locus as a supportive marker for forensic DNA profiling. Materials and methods: Amplification of the SE33 locus was performed using the PowerPlex ES Monoplex System SE33 (Promega). After genotyping 204 Pakistani individuals, different genetic and forensic parameters for the SE33 locus were studied. Results: Genotyping of the SE33 locus revealed a total of 43 alleles including 3 novel alleles. Significant values of different forensic and genetic parameters including power of discrimination, power of exclusion, and polymorphism information content were observed. Conclusions: Addition of the SE33 locus in forensic DNA profiling may help to produce conclusive reports where results are inconclusive due to degraded evidence samples. The SE33 locus can confidently be used for Pakistani and neighboring populations having common ancestors from Iran to Central Asia, the Middle East, India and Turkey.

Analysis of paternity exclusions in the material collected by the Department of Forensic Medicine, Medical University of Bialystok in 2008-2017.

AIM OF THE STUDY: Analysis of frequency and structure of paternity exclusions in the material collected by the Department of Forensic Medicine, Medical University of Bialystok in 2008-2017. MATERIAL AND METHODS: The paper is based on paternity test reports involving alleged father-child-mother trios. In a total of reviewed 958 cases, 187 exclusions were identified. The analysis was carried out on the basis of the results of DNA tests. DNA extraction was performed using QIAamp DNA Mini Kit (Qiagen) and DNA quantitation using Quantifiler Human DNA Quantification Kit and 7500 Real-Time PCR System (Applied Biosystems). AmpFLSTR Identifiler PCR Amplification Kit and a PCR System 9700 thermal cycler (Applied Biosystems) were used for DNA amplification. RESULTS: Over the analyzed period, the number of paternity tests was nearly halved, whereas the percentage of exclusions in individual years varied significantly (33.9-13.3%), with the average of 26.3%. The highest efficiency of exclusions was observed for D18S51 (0.7166) and FGA (0.7059), and the least effective system was TPOX (0.3048). CONCLUSIONS: The applied set of markers has been demonstrated to be an efficient tool in genetic paternity tests in the context of the recommended rules of exclusion.

Polymorphism of 15 short tandem repeat loci in Hui population of Ningxia Tongxin district.

OBJECTIVE: To investigate the polymorphism of 15 STR loci in Hui population from Ningxia Tongxin district. METHODS: Identifiler Plus kit used to detect the allelic frequencies of 15 STR loci in 598 unrelated Tongxin Hui individuals, and the population genetics parameters were calculated by using statistic software. RESULTS: The results demonstrated that all loci were found to be no deviation from Hardy-Weinberg equilibrium (P > 0.05), Heterozygosity (H) ranged from 0.637 to 0.868, matching probability (Pm) ranged from 0.034 to 0.213, power of discrimination (DP) ranged from 0.787 to 0.966, polymorphism information content (PIC) ranged from 0.560 to 0.850, power of exclusion (PE) ranged from 0.338 to 0.730. CONCLUSIONS: The 15 STR loci are relatively abundant in polymorphic information and suitable for paternity testing and personal identification.

DNA profiles from clothing fibers using direct PCR.

We report on the successful use of direct PCR amplification of single fibers from items of worn clothing. Items of clothing were worn throughout the course of a day, with the individual commencing regular activities. Single fibers were taken from the cuff of the clothing at regular intervals and amplified directly. The same areas were subjected to tape-lifting, and also amplified directly for comparison. The NGM™ kit that amplifies 15 STR loci plus amelogenin was used. A total of 35 single fiber samples were processed and analyzed from five items of clothing, with 81 % of samples returning a profile of 14 alleles or more. All tape-lift samples amplified directly produced DNA profiles of 15 alleles or more. The aim was to develop a simple, operational method that could be used routinely in forensic science casework and that has the potential to generate more complete profiles, which would not be detected using standard extraction methods on this type of sample. For ease of implementation, the process also adheres to standard methods with no increase in the cycle number.

STR allele sequence variation: Current knowledge and future issues.

This article reviews what is currently known about short tandem repeat (STR) allelic sequence variation in and around the twenty-four loci most commonly used throughout the world to perform forensic DNA investigations. These STR loci include D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, SE33, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045. All known reported variant alleles are compiled along with genomic information available from GenBank, dbSNP, and the 1000 Genomes Project. Supplementary files are included which provide annotated reference sequences for each STR locus, characterize genomic variation around the STR repeat region, and compare alleles present in currently available STR kit allelic ladders. Looking to the future, STR allele nomenclature options are discussed as they relate to next generation sequencing efforts underway.

Identification of the sequence variations of 15 autosomal STR loci in a Chinese population.

BACKGROUND: DNA sequence variation including base(s) changes and insertion or deletion in the primer binding region may cause a null allele and, if this changes the length of the amplified fragment out of the allelic ladder, off-ladder (OL) alleles may be detected. AIM: In order to provide accurate and reliable DNA evidence for forensic DNA analysis, it is essential to clarify sequence variations in prevalently used STR loci. SUBJECTS AND METHODS: Suspected null alleles and OL alleles of PlowerPlex16® System from 21,934 unrelated Chinese individuals were verified by alternative systems and sequenced. RESULTS: A total of 17 cases with null alleles were identified, including 12 kinds of point mutations in 16 cases and a 19-base deletion in one case. The total frequency of null alleles was 7.751 × 10(-4). Eight hundred and forty-four OL alleles classified as being of 97 different kinds were observed at 15 STR loci of the PowerPlex®16 system except vWA. All the frequencies of OL alleles were under 0.01. CONCLUSION: Null alleles should be confirmed by alternative primers and OL alleles should be named appropriately. Particular attention should be paid to sequence variation, since incorrect designation could lead to false conclusions.

Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow.

The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5ng, however, the optimum range determined was 2.5-10ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR(®) Identifiler(®) Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit.