Allelic Ladder Research Articles

The development of novel genome-SSRs, multiplex PCR panels, and allelic ladders for parentage identification in Tachypleus tridentatus

Tachypleus tridentatus, one of the oldest animal species on Earth, with significant biological and ecological value, its population sizes have declined due to overfishing. Stock enhancement technology has been used to protect T. tridentatus, however, its efficiency needs to be addressed. In the present study, a parentage identification system was developed. We identified 235,336 SSRs and 182,394 primer pairs were designed. Out of 321 synthesized primers, 14 highly polymorphic primers were selected to reveal the genetic diversity of 110 T. tridentatus. SSRs were amplified with the 14 developed primers, which were fluorescently labeled, and the resulting PCR products were analyzed using the ABI DNA Analyzer 3730xl. The number of alleles per locus, the polymorphism information content, the observed heterozygosity, and the expected heterozygosity ranged from 7 to 48, 0.581 to 0.963, 0.636 to 0.909, and 0.645 to 0.969, respectively. The cumulative exclusion probability with both unknown parents, with only unknown parents, or with both known parents was >99.9999% for the 14 loci. Furthermore, to facilitate data exchange across laboratories and platforms, multiplex PCR detection, SSR fragment size ladders, and parentage identification were employed. Out of the 14 primers, 6 optimal primers were selected for multiplex PCR validation, and two sets of triplex systems were established. Three thousand clones for 160 alleles of the six primers were selected for plasmid extraction and sequencing, and 600 correct plasmids were obtained for new T. tridentatus SSR fragment size ladder construction. Two pairs of parents with 12 offspring each were used for parentage identification by the multiplex PCR systems and ladders, the cumulative average exclusion probability of these 6 primers reached 99.9999% among parents and their offsprings, and was 0% in nonparent-offspring group. The parentage identification system constructed in this study can serve as a tool for monitoring the survival rates of T. tridentatus in stock enhancement programs.

Application of bioluminescence assay to assess PCR carryover contamination in forensic DNA laboratories

In forensic DNA testing, PCR-based multilocus short tandem repeat (STR) profiling kits, which have high sensitivity and discriminatory power, are generally used to analyze autosomal and Y-chromosomal DNA profiles. Forensic DNA laboratories require strict quality control for DNA testing, as contamination during analyses leads to incorrect interpretation of DNA profiles. Here, we aimed to apply bioluminescence assay to detect and monitor residual PCR products on laboratory work area and equipment surfaces by targeting dATP in the PCR product and allelic ladder marker. Two commercially available bioluminescence assay kits (CheckLite HS Plus and UltraSnap™) were examined for their sensitivity after confirming their reactivity to dATP. In the assay using CheckLite HS Plus, the lower detectable sample volumes were calculated as 10 pl of PCR product of GlobalFiler and PowerPlex Fusion and 1 pl of PCR product of Yfiler Plus and the allelic ladder marker of GlobalFiler, whereas those in the assay using UltraSnap™ were calculated as 1 nl of PCR product and allelic ladder marker. The sample volumes of these kits were lower than those detected through electrophoresis. Thus, the sensitivity of these kits was sufficient to control PCR carryover contamination in the post-PCR areas. Furthermore, residual PCR products in the post-PCR areas were continuously monitored using a bioluminescence assay. The results showed that the bioluminescence values increased after handling PCR samples for electrophoresis and decreased after decontamination. Therefore, we concluded that the bioluminescence assay is useful for assessing PCR carryover contamination in post-PCR processes in forensic DNA laboratories.

The distribution of allele frequencies of the 15 Short Tandem Repeats loci in Elaziğ population, Türkiye

The DNA within the Human Genome involves loci with high polymorphism such as short tandem repeats (STR). The STR polymorphism analysis is extensively used for numerous purposes such as identification studies in forensic sciences, paternity tests, genome mapping, and population studies. In this study, blood and swab samples were obtained from 200 unrelated healthy individuals living in Elazig province. DNA was amplified via the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, USA) to analyze the 15 STR loci. Using the ABI PRISM310 Genetic Analyzer capillary electrophoresis, the samples were analyzed and the allelic ladder was used to compare and identify the alleles of the samples, the distribution of the allele frequencies and peak values were examined. For statistical analysis Arlequin v.3.11 and PowerStats v.1.2 were used to assess the data. To determine if the allele frequencies were compatible with the Hardy-Weinberg (HW) equilibrium, the Markov chain and the Fisher's exact test p-value (p<0.05) were examined. It was determined that the D8S1179 (0.00002), D21S11 (0.00593) and vWA (0.04939) loci were not compatible with the HW equilibrium, but the rest of the loci were compatible. For the loci, the combined power of exclusion is 0.9999995, the combined power of discrimination is 0.9999999999998. The probability of matching for the 15 STR loci was calculated to be 1 in 1.364x10ˉ17. The power of discrimination was quite high when all the loci were considered. The results of this study indicate that the populations residing in the Elaziğ province may benefit from paternity testing, forensic identification, and genomic mapping using the data as an appropriate marker.

A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

An X-STRs analysis of the Iraqi Sorani Kurds.

A database for the Iraqi Sorani Kurds, specifically focused on the 12 X-short tandem repeat (STR) loci, has been developed to fascilitate forensic and population genetics investigations. The present study involved genotyping 117 unrelated individuals from the Sorani Kurds ethnic group using the Investigator Argus X-12 QS kit. The analysis revealed that the DXS10135 locus exhibited the highest degree of polymorphism, as indicated by a polymorphism information content (PIC) value of 0.94565 and a gene diversity (GD) value of 0.95623. Conversely, the DXS8378 locus displayed the lowest level of polymorphism, with a PIC value of 0.61026 and a GD value of 0.68170. Notably, two individuals were found to possess a rare allele (allele = 6) at the DXS8378 locus, which was not included in the allelic ladder of the kit. Furthermore, a significant linkage disequilibrium (LD) (p < 0.05/117) was observed between the DXS10103 and DXS10101 loci on linkage group 3 (LG3). The ancestral composition of the five primary geographic regions, namely Africa, Middle East, East Asia, Europe, and South America, was determined through the utilization of the [Formula: see text] ratio. The findings of this analysis revealed that the Middle Eastern populations exhibited the lowest [Formula: see text] ratio, measuring at 0.23243, indicating a relatively lower ancestral diversity. Conversely, the European populations showcased the highest [Formula: see text] ratio, measuring at 0.27122, indicative of a greater ancestral diversity within this region. Additionally, the allelic richness indicators, namely distinctive and private alleles, indicated that Africa and the Middle East displayed the highest levels, while Far East Asia exhibited the lowest. This analysis supports the hypothesis of repeated founder effects during outward migrations, as evidenced by both the ancestry variability and the allelic richness. Consequently, the findings of this study have important implications for forensic genetics and population genetics research, particularly in relation to the consideration of genetic predispositions within specific ethnic groups.

A preliminary exploration for co-detecting RNA virus and STR type on capillary electrophoresis in forensic practice.

RNA virus infection such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection shows severe respiratory symptoms on human and could be an obvious individual characteristic for investigations in forensic science. As for biological samples suspected to contain RNA virus in forensic casework, it requires respective detection of viral RNA and human DNA: reverse transcriptase polymerase chain reaction and DNA type (short tandem repeat [STR] analysis). Capillary electrophoresis (CE) has been shown to be a versatile technique and used for a variety of applications, so we preliminarily explored the co-detection of RNA virus and STR type on CE by developing a system of co-detecting SARS-CoV-2 and STR type under ensuring both the efficiency of forensic DNA analysis and safety of the laboratory. This study investigated the development and validation of the system, including N and ORF1ab primer designs, polymerase chain reaction amplification, allelic ladder, CE detection, thermal cycling parameters, concordance, sensitivity, species specificity, precision, and contrived and real SARS-CoV-2 sample studies. Final results showed the system could simultaneously detect SARS-CoV-2 and STR type, further indicating that CE has possibilities in the multi-detection of RNA viruses/STR type to help to prompt individual characteristics (viral infection) and narrow the scope of investigation in forensic science.

Validation study of 6 dye-based SF25™ PCR amplification kit

Ever since the inception of STR-based forensic DNA analysis for human identification and DNA profiling, the approach developed continuously. Most of these developments are pertaining to achieve the set of best polymorphic loci for analysis, high inhibitor tolerance capability, and increase in sensitivity, reduction in turnaround time, and cost-effectiveness. On the similar line, the SF25™ PCR amplification kit, a 6 fluorescent dye-chemistry based autosomal STR kit was developed, which analyses 25 STR loci viz., FAM fluorescent dye-labelled D3S1358, D13S317, D7S820, D16S539, D1S1656, and Penta E; HEX fluorescent dye-labelled TPOX, TH01, D2S1338, CSF1PO, and Penta D; SUM fluorescent dye-labelled D19S433, vWA, D21S11, D18S51, and D6S1043; LYN fluorescent dye-labelled Amelogenin, D8S1179, D5S818, D12S391, and FGA; PUR fluorescent dye-labelled D22S1045, Y-indel; D2S441 and D10S1248. The developmental validation of the SF25™ PCR Amplification reagents were carried out in this study to analyze its specificity, sensitivity, and concordance. This multiplex kit showed higher sensitivity with 125 pg of DNA template generating a complete profile (with 100% allele call). Out of 316 samples, 32samples showed aberrant triallelic pattern/aberrant heterozygous pattern at D16S539 locus. The possible discrepant homozygous condition at locus D1S1656 might be due to the invasion of alleles at D1S1656 locus present adjacent to D16S539 locus. Such invasion resulted due to the shortening of the allelic bin size at the D1S1656 locus. The allelic ladder of the SF25™ PCR amplification kit reveals the allelic range of D1S1656 from 11 to 19.3 which can be interpreted according to the casework as deemed by the end-users for their samples.

Detection of Main Causative Agents among Young Children Suffering from Epiglottitis in Hilla City, Iraq.

Epiglottitis is a rapidly progressive epiglottis infection leading to upper airway edema. This study aimed to detect the main causative agent, viral infection, by immunofluorescence antibody technique and PCR technique and bacterial infection detection by specific gene among young children suffering from epiglottitis. This study included 85 young children aged 10-15 years. The virus was identified on 85 blood samples using the CER test Human simplex virus Card test; the results revealed that 12 (14.1%) specimens were related to virus infection, and the sera of patients showed anti-IgM to HSV-1 antibodies. HSV-1 was detected in blood samples by qPCR technique. Eighty-five saliva samples were collected from young children suffering from epiglottitis. The samples were cultured for 18-24 hours at 37°C. They were then cultivated for 18-24 hours on various selective media at 37°C. The colony morphology, microscopically, and biochemical testing were used to identify Haemophilus influenzae as a first Identification. Out of 85 clinical specimens, 63 (74.1%) were positive culture, while 22 (25.9%) had no growth on culture media; out of 63 specimens, only 22 (34.9%) isolates belonged to Haemophilus influenzae by biochemical tests, while 41 (65.1%) related to other types of microorganisms. VITEK 2 was used to validate bacteria isolates from young children suffering from epiglottitis. The findings indicate that 22 (34.9%) isolates related to Haemophilus influenzae have been confirmed with an excellent ID message confidence level (94 to 99.8% likelihood percentage). This method is characterized by quick bacterial detection. DNA was taken from all suspected isolates previously identified as Haemophilus influenzae using the vitek2 technology, and traditional PCR was used to amplify specific hel gene for Haemophilus influenzae primers utilizing these DNA samples. After that, when compared to an allelic ladder, gel electrophoresis revealed that all 22 (100%) samples of Haemophilus influenzae produced 101 bp DNA fragments. For isolates previously identified as Haemophilus influenzae, molecular identification of the ompP gene was performed. The results showed that 12 (or 54.5 percent) of the 22 isolates tested positive for this virulence gene. When compared to an allelic ladder, the presence of (459 bp) bands indicated positive results. In addition, the bexA gene was molecularly detected in 22 Haemophilus influenzae isolates, showing that only 8 (36.3 percent) of the isolates had this gene. When compared to an allelic ladder, the presence of a (343 bp) band indicated positive results for bexA gene pathogenicity; in conclusion, HSV (1) and Hib were considered almost causative agents of epiglottitis in young children.

Design and development of novel single multiplex system incorporating 26 rapidly mutating Y-STRs; 26 RM Yplex.

This article details the development of a single multiplex system amplifying 26 rapidly mutating Y-STR markers. A sequenced allelic ladder, constructed for calling alleles of all loci, is introduced. The multiplex system shows the ability to address the limitations of Y-STRs commercial kits in differentiating closely related males. The multiplex performed well in the prevalidation tests and showed great potential to be used in forensic casework.

Genotypic assay to determine some virulence factors of Uropathogenic E. coli (UPEC) isolates

A total of 179 urine samples were collected from patients suffering from urinary tract infections were admitted and visit Al-Hilla General Teaching Hospital in Al-Hilla City, during a period from April 2021 to December 2021, from both sex (male and female). Out of 179,123 (68.7%) were positive culture, whereas 56 (31.3%) samples showed no bacterial growth, To confirm the identification of E. coli by use selective media (EMB agar medium, biochemical tests, automated Vitek 2 system and 16s RNA specific primer by the presence of (1492 bp) compared with allelic ladder, it was found that, E. coli were deliberated the main an etiological causes UTI to other types bacteria which constitute 56/123 (45.5%), [45/56 (80.4%) from female and 11/56 (19.6%) from male], while 67/123 (54.4%) were related to other types of bacteria. Molecular detection of some virulence factors genes were studied, out of 56 E. coli isolates, hlyA gene was detected in 21/56 (37.5%) isolates by the presence of (1177 bp) and sat gene was detected in 35/56 (62.5%) isolates by the presence of (410 bp) compared with allelic ladder.

Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses.

Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we found that good tetraSTRs do exist in horses but, as expected, at a much lower frequency than in other species, e.g., dogs and humans. Using a series of efficient methods developed in our laboratory for the production of multiplexed tetraSTRs in other species, we identified a set of tetra- and pentaSTRs that we developed into a 17-plex panel for the horse, plus a sex-identifying marker near the amelogenin gene. These markers were tested in 128 horses representing 16 breeds as well as crossbred horses, and we found that these markers have useful genetic variability. Average observed heterozygosities (Ho) ranged from 0.53 to 0.89 for the individual markers (0.66 average Ho for all markers), and 0.62-0.82 for expected heterozygosity (He) within breeds (0.72 average He for all markers). The probability of identity (PI) within breeds for which 10 or more samples were available was at least 1.1 x 10−11, and the PI among siblings (PIsib) was 1.5 x 10−5. Stutter was ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs. We predict that it will be possible to develop accurate allelic ladders for this multiplex panel that will make cross-laboratory comparisons easier and will also improve DNA profiling accuracy. Although we were only able to exclude candidate genes for Friesian horse megaesophagus with no unexcluded genes that are possibly causative at this point in time, the study helped us to refine the methods used to develop better tetraSTR multiplexed panels for species such as the horse that have a low frequency of tetraSTRs.

Genomic Analysis study of Virulence Potential α-hemolysin from Uropathogenic E.coli

Uropathogenic E.coli (UPEC) is the source of numerous toxins that cause nosocomial and community-acquired UTIs, including -hemolysin, a secreted lipoprotein that is a powerful and ubiquitous cytolysin that belongs to the (RTX) family. Between March 2021 to December 2021, 123urine samples were collected from patients with urinary tract infections who were admitted Al-Hilla General Teaching Hospital in Al-Hilla City, from both sexes (male and female). Out of (123), 56(45.5%) were positive culture for E. coli which identified by selective media, biochemical tests, Vitek 2 system and 16s RNA gene specific primer by the presence of (1492 bp) compared with allelic ladder, while other causes recorded 67(54.5%). DNA purification Kit supplemented by the manufacturing company Geneaid, (Korea). Primers were used and PCR condition to amplify sequences of the 16SrRNA and hlyA genes. Amplification products were separated by electrophoreses. The gene sequencing for 16SrRNA and HlyA gene was done by sanger sequencing method and phylogenic analysis. The study pointed to the number of mutation that happen with HlyA gene, as α-hemolysin gene varies greatly between local and international strains.

DNA databases of a CITES listed species Aquilaria malaccensis (Thymelaeaceae) as the tracking tools for forensic identification and chain of custody certification

Aquilaria malaccensis (Thymelaeaceae) is the main source of high-grade agarwood in Southeast Asia. Aggressive collections and trade activities over the past decades have put great pressure on the natural stands and raised concerns over the long-term survival potential of A. malaccensis. Tracking and authentication of agarwood require method with a high degree of accuracy. Therefore, this study aimed to develop DNA databases of A. malaccensis as the tracking tools at species, population and individual levels for forensic identification and chain of custody certification. Using two cpDNA (rbcL and matK) and an rDNA (ITS2) markers, species identification database of Aquilaria was developed to distinguish A. malaccensis from A. hirta, A. microcarpa, A. beccariana, A. crassna, A. sinensis and A. rostrata. In addition, based on 35 populations of A. malaccensis throughout Peninsular Malaysia, cpDNA haplotype and STR allele frequency databases were developed for population and individual identification. A haplotype distribution map based on 29 haplotypes derived from seven cpDNA showed that the A. malaccensis in Peninsular Malaysia can be associated to Kedah-Perak and Kelantan-Johor regions. Similarly, genetic relatedness and Bayesian clustering analyses based on 10 STR markers also divided the 35 populations into two main genetic clusters, corresponding to Kedah-Perak and Kelantan-Johor regions. The STR allele frequency databases were established and characterized according to these two regions. To determine the performance of the STR allele frequency databases for population identification, independent self-assignment tests showed that the percentage of individuals correctly assigned into the origin population was 93.88% in Kedah-Perak and 90.29% in Kelantan-Johor. For the STR allele frequency databases to be used for individual identification, conservativeness tests showed that the θ should be adjusted to 0.250 and 0.200 in the Kedah-Perak and Kelantan-Johor databases, respectively. To ensure consistency in allele calling for the dinucleotide repeat loci across different electrophoretic platforms or laboratories, allelic ladders have been developed for the 10 STR loci. Two case studies are presented of how these databases were used to track A. malaccensis to the origin population and stump. These databases are ready to be used to provide admissible forensic evidence for legal proceedings against the illegal harvesters of agarwood and for agarwood certification to meet the consumer country regulations.

STRoe deer: A validated forensic STR profiling system for the European roe deer (Capreolus capreolus)

European roe deer (Capreolus capreolus L.) are the most common game species in Europe, hunted for meat and trophies. Forensic investigations involving roe deer poaching may often benefit from an individual identification method to link a suspect to a specific incident. The current paper presents a forensically validated DNA profiling system for European roe deer called “STRoe deer”. This DNA profiling system consists of 12 novel unlinked tetra-nucleotide short tandem repeat (STR) loci and two sexing markers, with an allelic ladder to facilitate accurate genotyping. Validation results using 513 European roe deer samples collected from a single population from the Swiss Plateau demonstrated successful amplification of all 14 loci with as little as 0.05 ng of European roe deer DNA. Species-specificity tests showed that other members of the Cervidae family exhibited partial profiles and non-specific peaks, whereas most members of the Bovidae family showed just non-specific cross-species amplification products. Three different methods to calculate match probabilities for randomly sampled European roe deer genotypes resulted in median match probabilities ranging from 1.4 × 10−13 to 2.5 × 10−5. These methods accounted for possible population structure, occurrence of null alleles and individual relatedness. Based on these results, we conclude that STRoe deer is a robust genotyping system that should prove a valuable tool for individual identification and sexing of European roe deer to support criminal investigations.

OdoPlex: An STR multiplex panel optimized and validated for forensic identification and sex determination of North American mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus)

OdoPlex is a single 5-dye multiplex panel for forensic identification of North American mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus). This robust panel is comprised of 15 autosomal markers (14 tetranucleotide loci and a single dinucleotide locus) and two sex-linked markers. OdoPlex was optimized and validated in response to the USA-based Organization of Scientific Area Committees (OSAC) research priority calling for the development and validation of standardized STR panels and associated allelic ladders for North American species routinely encountered in wildlife forensic casework. Genotypes were obtained from a combined 1143 white-tailed deer and mule deer representing 18 subspecies, including both the Key deer and Columbian white-tailed deer, two ESA-listed subspecies of white-tailed deer of special interest to population managers. Complete STR genotypes, including sex identification, were generated from as little as 125 pg of DNA template. Methods for species-level assignment and the calculation of single-source likelihood ratios accounting for population substructure are discussed in detail. Median likelihood ratios ranged from 1.65 × 1010 (O. hemionus) to 1.58 × 1014 (O. virginianus), making OdoPlex a powerful assay for individual identification of deer in the genus Odocoileus.

Construction of an in-house allelic ladder for Canine Genotypes™ Panel 2.1 Kit.

Dogs (Canis lupus familiaris) are among the most common companion animals in the Republic of Korea. Recently, there have been many criminal cases of dog cruelty, injury, and theft, among others. This has increased the importance of dog-related biological evidence at crime scenes. The National Forensic Service of the Republic of Korea conducts short tandem repeat (STR) analysis using the Thermo Scientific Canine Genotypes™ Panel 2.1 Kit (Canine Kit) to identify individual dogs through forensic analysis. The Canine Kit was developed as a forensic STR kit for the identification of individual dogs. However, an allelic ladder was neither developed nor included in the commercial kit, leaving an issue of accurate genotyping. Primer details for the 18 markers used in the Canine Kit are proprietary information, and thus, unavailable to end-users. In this study, an allelic ladder was constructed with 160 fragments by combining 158 fragments of STR alleles obtained by nested PCR and two fragments artificially obtained from the sex-determination marker. By including the new allelic ladder in analysis of samples amplified with the Canine Kit, the accuracy and reliability of data analysis were improved. Application of this allelic ladder would be helpful for interlaboratory data sharing and standardization of canine genotype databases.

OpiumPlex is a novel microsatellite system for profiling opium poppy (Papaver somniferum L.)

Opium poppy (Papaver somniferum L.) is a versatile plant exploited by the pharmaceutical and food industries. Unfortunately, it is also infamously known as a source of highly addictive narcotics, primarily heroin. Drug abuse has devastating consequences for users and also has many direct or indirect negative impacts on human society as a whole. Therefore, developing a molecular genetic tool for the individualization of opium poppy, raw opium or heroin samples could help in the fight against the drug trade by retrieving more information about the source of narcotics and linking isolated criminal cases. Bioinformatic analysis provided insight into the distribution, density and other characteristics of roughly 150 thousand microsatellite loci within the poppy genome and indicated underrepresentation of microsatellites with the desired attributes. Despite this fact, 27 polymorphic STR markers, divided into three multiplexed assays, were developed in this work. Internal validation confirmed species-specific amplification, showed that the optimal amount of DNA is within the range of 0.625–1.25 ng per reaction, and indicate relatively well balanced assays according to the metrics used. Moreover, the stutter ratio (mean + 3 SD 2.28–15.59%) and allele-specific stutters were described. The analysis of 187 individual samples led to the identification of 158 alleles in total, with a mean of 5.85 alleles and a range of 3–14 alleles per locus. Most of the alleles (151) were sequenced by the Sanger method, which enabled us to propose standardized nomenclature and create three allelic ladders. The OpiumPlex system discriminates most of the varieties from each other and pharmaceutical varieties from the others (culinary, dual and ornamental).

لا يوجد عنوان بالعربي في ملف الطلب على النظام

Background: The relationship testing through DNA profiling may undesirably be affected by the rare allele variants, tri-allelic pattern and null alleles. Therefore, it is vital to report such anomalies. We report a paternity testing in a sexual assault case studied at Punjab Forensic Science Agency, Lahore Pakistan showing a unique allele variant in mother and child. Methods: DNA was extracted from the buccal swabs of reference samples using organic extraction method and DNA profiling was done for 15 autosomal STRs and amelogenin using Identifiler Plus kit. Results: A novel out of marker range (OMR) allele variant between STR Loci D16S539 and D2S1338 was observed in the DNA profiles of victim (mother) as well as the child. At STR locus D2S1338 Twenty one different allele variant are listed at STRBase ranging from 11 to 28. The allele variant observed in this case study was appeared at less than marker range (&lt; D2S1338) with a size of 297.50 bp. The novel variant OMR allele at D2S1338 was labeled as allele 13, when compared to the other allele in allelic ladder. Moreover, the PFSA DNA database was searched for this unique allelic variation and it was found that this was present in only two other samples of distinct cases. Conclusion: The overall frequency of this unique allele variant was 3 in 10,125 unrelated individuals with frequency of occurrence of 0.0296. According to our limited knowledge it is the first report of a novel OMR allele variant at D2S1338 in Pakistani Population.

Genetic polymorphisms of 21 non-combined of DNA index system short tandem repeat loci in Hainan Li population

To investigate the genetic polymorphisms of 21 non-combined DNA index system short tandem repeat (STR) loci in Hainan Li population. DNA samples from 339 unrelated healthy individuals of Li population from Hainan Province were extracted and amplified with fluorescence labeled multiplex PCR system. PCR products were electrophoresed on an ABI3130 Genetic Analyzer following the manufacturer's instructions. Allele designation was performed with a GeneMapper ID-X by comparison with the allele ladder provided by the corresponding kit. A total of 173 alleles and 489 genotypes were observed for the 21 STR loci, respectively. The frequencies of alleles and genotypes were 0.0010-0.5434 and 0.0020-0.3274, respectively. The heterozygosity varied from 0.639 to 0.833. Discrimination power (DP) was 0.803-0.948, power of exclusion for trio-paternity was 0.416-0.584, power of exclusion for duo-paternity was 0.140-0.238, the polymorphism information content(PIC) was 0.57-0.81, respectively. The total discrimination power (TDP), cumulative probability of exclusion for trio-paternity testing(CPE-trio) and cumulative probability of exclusion for duo-paternity testing (CPE-duo) were 0.999 999 999 999 99, 0.999 999 883 211 752, and 0.987 266, respectively. The 21 STR loci are highly polymorphic and informative in the studied population and can be employed as supplementary loci in duo-paternity testing or cases with variant circumstances.

An inter-laboratory study of DNA-based identity, parentage and species testing in animal forensic genetics

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.