Genetic variation among the major Pakistani populations based on 15 autosomal STR markers.

Pakistan is located at an important cross-road of human history and has been a passageway for many invaders and dynasties in the past. The historic human migrations across this country have resulted in a blend of ancient civilizations, which are still reflected in the current socio-cultural fabrication of this population. This makes Pakistan an ideal country to study the genetic differentiation and various other genomic aspects of a human population.

Comparative performance of AmpFLSTR® Identifiler® Plus PCR amplification kit and QIAGEN® Investigator® IDplex Plus kit.

Many forensic STR kits are currently available in the market. The AmpFLSTR® Identifiler® Plus kit, which targets 15 STRs, is commonly used worldwide. The Thai forensic DNA community is built around it in terms of instrument, databases, and interpretation. QIAGEN's IDplex Plus kit targets the same loci, but the PCR cycling time is shorter by about 90min. A direct comparison that assesses forensic parameters and applicability to casework between the two kits has never been carried out. In this study, we performed a direct comparison between the two kits using serial dilutions of two control DNA samples and 60 randomly selected casework samples, including samples taken from improvised explosive devices and terrorist raids. We statistically compared the performance of the two kits in terms of peak height, number of allele detected (allelic drop-out), intra-locus balance, inter-locus balance, inhibitor tolerance, stutter ratio, concordance, and allelic drop-in. The results demonstrate that both kits are statistically similar in performance. IDplex Plus gave higher peak heights in sensitivity test and tolerated inhibitors better, but had slightly worse inter-locus balances and stutter ratios. However, these differences were not practically significant, as seen by the resulting profiles of the casework samples (p=0.601). The performance on low-template samples also was not different. In conclusion, laboratories looking to replace the aging Identifiler® Plus might consider the IDplex Plus as a faster, more robust alternative that fits right into their existing structure without further investment in instrument and DNA database. Having more kits available worldwide by different companies could help bring the technology to different forensic laboratories and the justice system as a whole.

Forensic DNA typing strategy of degraded DNA on discarded cigarette ends using the AmpFℓSTR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR amplification kits.

DNA left on a forensic sample is often prone to degradation, especially if left to the elements. To maximize the chance of retrieving the most information from such compromised DNA, an appropriate profiling scheme using the available technologies needs to be devised. In this study, a total of 62 cigarette ends collected under different conditions of environmental exposure were employed to test the effectiveness of three DNA amplification kits, namely the Applied Biosystems™ AmpFℓSTR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR Amplification Kits, in the profiling of such compromised DNA. We demonstrated that Identifiler® Plus could substitute Identifiler® to improve the effectiveness of profiling for those inhibited cigarette samples. MiniFiler™, on the other hand, could supplement Identifiler®/Identifiler® Plus profiles and provide additional genetic information to enhance the evidential value of the samples, especially for those that have suffered from DNA degradation to a greater extent. The findings in this work allowed us to propose a DNA profiling strategy as follow: 1) samples yielding complete Identifiler®/Identifiler® Plus profiles require no further testing with MiniFiler™; 2) samples yielding partial single-source profiles to be tested with MiniFiler™ to add genetic information; 3) samples yielding no results are unlikely to yield any results with MiniFiler™.

Forensic and phylogenetic characterization of 15 autosomal STRs in Hazara population of Pakistan.

In the current study, 217 unrelated individuals of the Hazara population were genotyped for 15 autosomal short tandem repeats to generate parentage and forensic efficacy parameters. Hazaras belong to the Shi'a sect and are recognized by their Turko-Mogholi features. We found that D2S1338 was the most discriminatory locus with a maximum power of exclusion and high value of polymorphism information content. Whilst the Combined Power of Discrimination (CPD), Combined Matching Probability (CMP) and Combined Power of Exclusion (CPE) were 0.999999999999999, 2.76796338879E-17 and 0.999999040733479 respectively. Furthermore, the pattern of genetic affinity with genetically assumed related populations was demonstrated through Heat Map and Phylogenetic analysis, which revealed a great level of genetic closeness of Hazaras with Mongol population and descendants of Genghis Khan. The resulting data can be used for forensic applications and anthropological studies.

Detection of a new allele variation in Chinese Han population at the STR locus SE33 (ACTBP2).

In order to apply a useful STR system in DNA database construction, we performed a population study in China. Allele and genotype frequencies for STR SE33 were obtained for a sample of 213 random Chinese in view of application in personal identification. And we observed a new structural variation of 21.2 allele at SE33 locus which is described here for the first time.

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.

A selection guide for the new generation 6-dye DNA profiling systems.

The Federal Bureau of Investigation (FBI) has recently expanded the CODIS core loci from the existing 13 to 20 as a new guideline, and laboratories from the US are required to comply with the new regulation before uploading or conducting identity search in the national database. The expanded CODIS format, which shares all the core loci commonly used in the European countries and the US, not only increases international compatibility, but also reduces the number of adventitious matches, and hence facilitates international law enforcement and counterterrorism endeavours. Here, we review the key performance measures of three new STR amplification systems with 6-dye chemistry, namely, the Investigator 24plex QS Kit from QIAGEN, the GlobalFiler™ PCR Amplification Kit from Applied Biosystems™, and the PowerPlex® Fusion 6C System from Promega. Our results have demonstrated that GlobalFiler displays the highest sensitivity among the tested kits, whereas Investigator 24plex shows a higher tolerance to common PCR inhibitors including Humic acid and Tannic acid. GlobalFiler and Fusion 6C, on the other hand, yield DNA profiles with better heterozygous peak height and intra-colour signal balance. Both GlobalFiler and Investigator 24plex exhibit slightly higher sensitivity than Fusion 6C in the profiling of minor components in DNA mixture, but the latter displays a higher consistency in the preservation of the mixture ratio. In summary, our work has demonstrated that these three profiling systems have their different performance features, and hence it is recommended that laboratories should select the most suitable kits according to their own requirements and operational needs.

Analysis of 17 STR data on 5362 southern Portuguese individuals-an update on reference database.

The main objective of this work consisted of the updating of allele frequencies and other relevant forensic parameters for the 17 autosomal STR loci provided by the combination of the two types of kits used routinely in our laboratory casework: AmpF/STR Identifiler(®) and the Powerplex(®) 16 Systems. This aim was of significant importance, given that the last study on these kits within the southern Portuguese population dates back to 2006, and, as a consequence, it was necessary to correct the deviation caused by population evolution over the last ten years so that they might be better applied to our forensic casework. For this reason genetic data from 5362 unrelated Caucasian Portuguese individuals from the south of Portugal who were involved in paternity testing casework from 2005 to 2014 was used. Of all the markers, TPOX proved to be the least polymorphic, and Penta E the most. Secondly, this up-to-date southern Portuguese population was compared not only with the northern and central Portuguese populations, but also with that of southern Portugal in 2006, along with populations from Spain, Italy, Greece, Romania, Morocco, Angola and Korea in order to infer information about the relatedness of these respective populations, and the variation of the southern Portuguese population over time.

Allele frequency data for 15 autosomal STR loci in eight Indonesian subpopulations.

Evolutionary and cultural history can affect the genetic characteristics of a population and influences the frequency of different variants at a particular genetic marker (allele frequency). These characteristics directly influence the strength of forensic DNA evidence and make the availability of suitable allele frequency information for every discrete country or jurisdiction highly relevant. Population sub-structure within Indonesia has not been well characterised but should be expected given the complex geographical, linguistic and cultural architecture of the Indonesian population. Here we use forensic short tandem repeat (STR) markers to identify a number of distinct genetic subpopulations within Indonesia and calculate appropriate population sub-structure correction factors. This data represents the most comprehensive investigation of population sub-structure within Indonesia to date using these markers. The results demonstrate that significant sub-structure is present within the Indonesian population and must be accounted for using island specific allele frequencies and corresponding sub-structure correction factors in the calculation of forensic DNA match statistics.

Could Secondary DNA Transfer Falsely Place Someone at the Scene of a Crime?

The occurrence of secondary DNA transfer has been previously established. However, the transfer of DNA through an intermediary has not been revisited with more sensitive current technologies implemented to increase the likelihood of obtaining results from low-template/low-quality samples. This study evaluated whether this increased sensitivity could lead to the detection of interpretable secondary DNA transfer profiles. After two minutes of hand to hand contact, participants immediately handled assigned knives. Swabbings of the knives with detectable amounts of DNA were amplified with the Identifiler(®) Plus Amplification Kit and injected on a 3130xl. DNA typing results indicated that secondary DNA transfer was detected in 85% of the samples. In five samples, the secondary contributor was either the only contributor or the major contributor identified despite never coming into direct contact with the knife. This study demonstrates the risk of assuming that DNA recovered from an object resulted from direct contact.

Sibling assessment based on likelihood ratio and total number of shared alleles using 21 short tandem repeat loci included in the GlobalFiler™ kit.

Sibling assessment using the 15 autosomal short tandem repeat (STR) loci included in the Identifiler® kit can be difficult when comparing an unidentified party to an alleged sibling. Therefore, we investigated the likelihood ratio (LR) and the total number of shared alleles (TNSA) for sibship determination using the 21 autosomal STR loci included in the GlobalFiler™ kit. We computationally generated the genotypes of 10,000 sibling pairs and 10,000 unrelated pairs based on previously reported allele frequencies of the 15 Identifiler loci and the remaining 6 GlobalFiler loci. The LR and the TNSA were then calculated in each pair using the 15 and 21 loci. Next, these calculations were applied to 22 actual sibling pairs. LR values ⩾ 10,000 were observed in 48% of the sibling pairs using the 15 loci and in 80% of the sibling pairs using the 21 loci. The TNSA distribution between siblings and unrelated pairs was more divergent in GlobalFiler than in Identifiler. TNSA values ⩾ 20 were found only in true siblings in Identifiler, while TNSA values ⩾24 in GlobalFiler. In Identifiler, all pairs with TNSA ⩾ 24 had LR values ⩾ 10,000 and the same was true in GlobalFiler for TNSA ⩾29. Therefore, increasing the number of loci is very efficient for sibship determination. The LR is most reliable for determining sibship. However, TNSA values may be useful for the preliminary method of LR values because LR value demonstrated a significantly positive correlation with TNSA value in both Identifiler and GlobalFiler.

A reference frequency database of 15 autosomal STRs in Chile.

We estimated the allele frequencies for the 15 autosomal STR loci included in the AmpFlSTR(®) Identifiler (Applied Biosystems, USA) in a sample of 986 unrelated non-Native American individuals collected at five different localities from Chile, namely, Iquique, Santiago, Concepción, Temuco and Punta Arenas. Frequency distributions and several forensic parameters were estimated at each recruitment site. In addition, analyses were carried out merging the data into five sample locations. No significant statistical differences could be detected between different regions in Chile. These data represent one of the very few studies performed on autosomal STRs in Chile and therefore provide a useful tool for forensic casework carried out in the country.

Population genetic data for 15 autosomal STR markers in Turkish Cypriots from Cyprus.

Fifteen autosomal short tandem repeat (STR) markers [D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA] were analyzed in 501 unrelated, randomly selected Turkish Cypriot individuals from the island of Cyprus. While no locus duplications or null alleles were detected in these samples, eight allelic variants were observed in total, 75% of which were intermediate allelic variants that were absent in the system allelic ladder. Allelic frequencies and statistical parameters of forensic interest were calculated at each locus. For the 15 STR loci tested, combined matching probability (pM) was 2.15717 × 10(-18) and combined power of exclusion (PE) was 0.9999995213. No deviations from the Hardy-Weinberg equilibrium were observed, except for the vWA locus, which became insignificant after the Bonferroni correction for multiple testing. Locus-by-locus comparisons of the Turkish Cypriot allelic frequencies with those published for the neighboring and/or historically related populations with similar loci coverage (Turkish, Greek, Greek Cypriot, Italian and Lebanese) revealed some statistically significant differences at one to five loci. In general, an increase in the number of such significant differences between the Turkish Cypriot data and those for other populations correlated closely with an increase in the geographic distance and/or a decrease in the amount of historical contact. The Turkish Cypriot autosomal STR population study will find immediate use in the Committee on Missing Persons in Cyprus Project on the "Exhumation, Identification and Return of Remains of Missing Persons" and it will also be available for criminal, parentage and other missing person investigations.

Relationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples.

The relationships between DNA degradation ratios and the number of detected loci were explored in extremely old seminal stains evaluated using three short tandem repeat (STR) kits: the AmpFlSTR® Identifiler™ PCR Amplification Kit (Identifiler), the AmpFlSTR® Yfiler™ PCR Amplification Kit (Yfiler), and the AmpFlSTR® MiniFiler™ PCR Amplification Kit (MiniFiler). DNA degradation ratios based on 41, 129, and 305bp DNA fragments were calculated (129:41 and 305:41), and the relationships between the ratios and storage duration were also explored. Using the Identifiler kit, the number of loci detected was strongly correlated with the 129:41 ratio (r=0.887), whereas the correlation with the 305:41 ratio was moderate (r=0.656). Using the Yfiler kit, the DYS385 amplicon was detected in all samples, suggesting that DYS385 may be resistant to degradation. The number of detected loci was strongly correlated with the 129:41 ratio (r=0.768), and moderately so with the 305:41 ratio (r=0.515). MiniFiler detected at least seven loci in all samples. In samples that did not yield full profiles, the undetected loci were D7S820 and D21S11, or D21S11 only, suggesting that these loci might be easily degraded. The number of loci detected using STR kits correlated with the DNA degradation ratios. In particular, the 129:41 ratio was particularly useful for estimating the number of loci detectable by STR kits. On the other hand, we suggest that storage duration cannot be accurately estimated using DNA degradation ratios; these ratios were not strongly correlated with storage duration (129:41; r=-0.698, 305:41; r=-0.550). However, the ratios may allow the identification of samples that have been stored for more than 40years.

An Accelerated Analytical Process for the Development of STR Profiles for Casework Samples.

Significant efforts are being devoted to the development of methods enabling rapid generation of short tandem repeat (STR) profiles in order to reduce turnaround times for the delivery of human identification results from biological evidence. Some of the proposed solutions are still costly and low throughput. This study describes the optimization of an analytical process enabling the generation of complete STR profiles (single-source or mixed profiles) for human identification in approximately 5 h. This accelerated process uses currently available reagents and standard laboratory equipment. It includes a 30-min lysis step, a 27-min DNA extraction using the Promega Maxwell(®) 16 System, DNA quantification in <1 h using the Qiagen Investigator(®) Quantiplex HYres kit, fast amplification (<26 min) of the loci included in AmpFℓSTR(®) Identifiler(®), and analysis of the profiles on the 3500-series Genetic Analyzer. This combination of fast individual steps produces high-quality profiling results and offers a cost-effective alternative approach to rapid DNA analysis.

STR profiling of epithelial cells identified by X/Y-FISH labelling and laser microdissection using standard and elevated PCR conditions.

During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described.

Population data for 15 autosomal STR loci in the Miao ethnic minority from Guizhou Province, Southwest China.

Fifteen autosomal short tandem repeat (STR) loci were genotyped using the AmpFISTR Identifiler PCR Amplification kit from 505 unrelated healthy individuals of Miao ethnic minority living in the Guizhou Province, Southwest China. Also, the genetic distance between Miao and nine related populations was compared.

Generation of DNA profiles from fingerprints developed with columnar thin film technique.

Partial-bloody fingerprints and partial fingerprints with saliva are often encountered at crime scenes, potentially enabling the combination of fingerprint and DNA analyses for absolute identification, provided that the development technique for fingerprint analysis does not inhibit DNA analysis. 36 partial-bloody fingerprints and 30 fingerprints wetted with saliva, all deposited on brass, were first developed using the columnar-thin-film (CTF) technique and then subjected to short tandem repeat (STR) DNA analysis. Equal numbers of samples were subjected to the same DNA analysis without development. Tris (8-hydroxyquinolinato) aluminum, or Alq3, was evaporated to deposit CTFs for development of the prints. DNA was extracted from all 132 samples, quantified, and amplified with AmpFlSTR(®) Identifiler Plus Amplification Kit. Additionally, DNA analyses were conducted on four blood smears on un-fingerprinted brass that had been subjected to CTF deposition and four blood smears on un-fingerprinted brass that had not been subjected to CTF deposition. Complete and concordant autosomal STR profiles of the same quality were obtained from both undeveloped and CTF-developed fingerprints, indicating that CTF development of fingerprints preserves DNA and does not inhibit subsequent DNA analysis. Even when there were no fingerprints, CTF deposition did not lead to inhibition of DNA analysis.

Application of Short Tandem Repeat markers in diagnosis of chromosomal aneuploidies and forensic DNA investigation in Pakistan.

PURPOSE: Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular diagnostics and molecular genetics research. AmpFlSTR® Identifiler™ PCR amplification kit is a multiplex system for co-amplification of 15 STR markers used worldwide in forensic investigations. This study attempts to assess forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies. METHODOLOGY: Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for forensic parameters using Identifiler STRs and 74 patients' samples with different aneuploidies were evaluated for diagnostic strengths of these markers. RESULTS: All STRs hold sufficient forensic applicability in Pakistani population with paternity index between 1.5 and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power ≥0.9 (except TPOX locus). Variation from Hardy-Weinberg equilibrium was observed at some loci reflecting selective breeding and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using Identifiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients. CONCLUSION: The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick and simultaneous identification of some common trisomic conditions. Adding more sex chromosome specific STR markers can immensely increase the diagnostic and forensic potential of this system.

Comparative performance between "next generation" multiplex systems and the new European Standard Set of STR markers in the Portuguese Population.

Various multiplex STR systems have been developed by the major commercial companies in the forensic genetics field to comply with the recent establishment of the global European Standard Set (ESS) of markers. Of the various alternatives available, our laboratory decided to test the recent ESSplex Plus system (Qiagen) and the NGM kit (Life Technologies), which share the same 15 STR loci and comprise the most recently established ESS markers (D1S1656, D2S441, D10S1248, D12S391 and D22S1045). Apart from evaluating the kits' technical performances, a population and segregation study was carried out on a Portuguese sample, with the aim of introducing the ESS markers in routine forensic casework. A total of 370 individuals were sampled for this purpose, comprising 120 true trios (125 fathers, 125 mothers and 120 sons/daughters). No deviations from Hardy-Weinberg equilibrium were detected for the five new loci in the Portuguese population and no genotyping inconsistencies were observed between kits. Parameters of forensic interest revealed that, of the five ESS markers, D1S1656 was the most informative in our sample. Comparison of performances between all autosomal multiplex systems available in our laboratory (ESSplex Plus, NGM, Identifiler Plus and Powerplex 16 HS), revealed that the multiplex kits with the ESS markers generally showed better performances and, among these, the ESSplex Plus kit showed slightly higher sensitivity and a better detection of degraded DNA information.