Fusion 6C System Research Articles

Forensic Characterization, Genomic Variability and Ancestry Analysis of Six Populations from Odisha Using mtDNA SNPs and Autosomal STRs.

Located on India's eastern coast, Odisha is known for its diverse tribes and castes. In the early days of genome sequencing technology, researchers primarily studied the Austroasiatic communities inhabiting this region to reconstruct the ancient origins and dispersal of this broad linguistic group. However, current research has shifted towards identifying population and individual-specific genome variation for forensic applications. This study aims to analyze the forensic efficiency and ancestry of six populations from Odisha. We assessed the SF mtDNA-SNP60™ PCR Amplification Kit by comparing it with PowerPlex® Fusion 6C System, a widely used autosomal STR (aSTR) kit, in an Indian cohort. Although the mtDNA SNP kit showed low discriminating power for individuals of a diverse population, it could identify deep lineage divergence. Also, we utilized mitochondrial and autosomal variation information to analyze the ancestry of six endogamous ethnic groups in Odisha. We observe two extremities-populations with higher West Asian affinity and those with East Asian affinity. This observation is in congruence with the existing information of their tribal and non-tribal affiliation. When compared with neighbouring populations from Central and Eastern India, multivariate analysis showed that the Brahmins clustered separately or with the Gopala, Kaibarta appeared as an intermediate, Pana and Kandha clustered with the Gonds, and Savara with the Munda tribes. Our findings indicate significant deep lineage stratification in the ethnic populations of Odisha and a gene flow from West and East Asia. The artefacts of unique deep lineage in such a diverse population will help in improving forensic identification. In addition, we conclude that the SF mtDNA-SNP60 PCR Amplification Kit may be used only as a supplementary tool for forensic analysis.

Analysis of 26 STR loci (PowerPlex® Fusion 6C System) in a mestizo population from Mexico city.

Short tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes to enable laboratories to use these genetic systems to interpret and solve forensic casework. However, in Mexico, there are no studies with the PowerPlex Fusion 6C System, which includes 26 STRs (23 autosomal STRs and 3 Y-STRs). 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 312 different alleles were observed. Combined PE and PD were 99.999999809866% and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations. The results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied and justify its possible application in other Mexican Mestizo populations.

Forensic Validation and Application Evaluation of IDentifier DNA Typing Kit (Yan-Huang34).

To investigate the technical performance of IDentifier DNA typing kit (YanHuang34) and evaluate its forensic application value. Following the Criterion of Forensic Science Human Fluorescence STR Multiplex Amplification Reagent (GB/T 37226-2018), IDentifier DNA typing kit (YanHuang34) was verified in 11 aspects of species specificity, veracity, sensibility, adaptability, inhibitor tolerance, consistency, balance, reaction condition verification, mixed samples, stability and inter batch consistency. The system efficiency of IDentifier DNA typing kit (YanHuang34) was compared with the PowerPlex® Fusion 6C System, VersaPlex® 27PY System and VeriFilerTM Plus PCR Amplification Kit. The IDentifier DNA typing kit (YanHuang34) was used to detect the swabs of biological samples in daily cases and the STR performances were observed. IDentifier DNA typing kit (YanHuang34) had good species specificity, veracity, adaptability, inhibitor tolerance and balance. The sensibility was up to 0.062 5 ng. It was able to detect different types of samples, degraded samples and inhibitor mixed samples. Complete DNA typing could be obtained for samples with the mixture ratio less than 4∶1. The system efficiency of IDentifier DNA typing kit (YanHuang34) was very high, with TDP up to 1-1.08×10-37, CPEtrio and CPEduo up to 1-5.47×10-14 and 1-6.43×10-9, respectively. For the touched biological samples in actual cases, the effective detection rate was 21.05%. The system efficiency of kinship, single parent and full sibling identifications was effectively improved. The IDentifier DNA typing kit (YanHuang34) is adaptive to the GB/T 37226-2018 requirements. It can be used for individual identification and paternity identification, and is suitable for application in the field of forensic science.

Transfer, persistence and recovery of DNA and mRNA vaginal mucosa markers after intimate and social contact with Bayesian network analysis for activity level reporting

In sexual assault cases, it can be challenging to identify the type of body fluids/ cell types present in a crime scene sample, especially the origin of epithelial cells. Therefore, more labs are applying mRNA body fluid analysis for saliva, skin and vaginal mucosa markers. To address activity level propositions, it is necessary to assign probabilities of transfer, persistence, prevalence and recovery of DNA and mRNA markers. In this study we analysed 158 samples (fingernail swabs, penile swabs and boxershorts) from 12 couples collected at different time points post intimate contact and after non-intimate contact in order to detect DNA from the person of interest (POI) and mRNA vaginal mucosa markers. Samples were DNA and RNA co-extracted and analysed with PowerPlex®Fusion 6C System and 19-plex mRNA primer mix respectively, using Endpoint PCR and the CE platform. Vaginal mucosa was detected up to 36 h post intimate contact, but also detected in one non-intimate contact sample. In 94% of intimate contact and 50 % of non-intimate contact samples the DNA results support the proposition that POI is the donor (LR ≥ 10,000). There was a strong association between the detection of vaginal mucosa and the average RFU value of the POI. The data were used to instantiate a comprehensive Bayesian network to evaluate the evidence at activity level, given alternate propositions conditioned upon indirect or direct transfer events. It is shown that the value of the evidence is mainly affected by the high DNA quantity (measured as mean RFU) that is recovered from the POI. The detection of vaginal mucosa had low impact upon the resultant likelihood ratio.

Allelic frequencies distribution and forensic parameters of 23 autosomal short tandem repeats in the population of the State of Pernambuco, Brazil

Located in the Northeast Region, the Pernambuco State is one of the 27 federative units of Brazil. Here, we determined populational data for 23 short tandem repeat (STR) markers – CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, PENTA D, PENTA E and SE33 — of the Pernambuco population. The sample consisted of 767 healthy, adult, unrelated individuals (437 males, 330 females) born and resident in the State of Pernambuco. STRs were amplified using three multiplex kits, according to the availability: PowerPlex® Fusion 6C System (Promega Corporation), PowerPlex® Fusion System (Promega Corporation) and GlobalFiler™ Express (Thermo Fisher Scientific). Allelic frequencies, forensic parameters and Hardy-Weinberg equilibrium determinations were estimated for all the 23 loci. No deviations from the Hardy-Weinberg equilibrium were observed for any of the markers, after Bonferroni correction. We observed that the most and less informative markers were SE33 and TPOX, respectively. The combined power of discrimination (CPD) was 0.99999999999999999999999999999, and the combined power of exclusion (CPE) was 0.99999999997. The cumulative typical paternity index was 37,919,301,869.3021. Interpopulation analyses (Nei’s genetic distance) based on the expanded CODIS core loci was performed between the Pernambuco sample and other global populations. Pernambuco was the closest Brazilian population to African group and stayed distant from the Native American group. This work contributed to show that a panel of 23 autosomal STR loci is very informative, being able for forensic applications related in this population.

Comparison of the Efficiency of GlobalfilerTM IQC PCR Amplification Kit and Powerplex® Fusion 6C System with Half-Volume Reactions for Forensic Trace DNA Testing

Two major challenges in daily and routinely forensic genetics test are samples with low or very low quantitiesof DNA and high cost of processing. To overcome these obstacles, a reduction in reaction volume to half wouldoffer a substantial benefit, but even so it should provide complete genetic profiles without conceding the qualityof the results. The aim of this study is to test the robustness of two commercially available kits, GlobalFilerTMIQC PCR Amplification Kit (GF-IQC) and PowerPlex® Fusion 6C System (P-6C), with half volume reaction inaccordance with SGWDAM guidelines. GF-IQC showed minimum and stochastic threshold of 45 and 610 RFU,while P-6C demonstrated 60 and 670 RFU of those values respectively. The P-6C can tolerate to many inhibitorsincluding melanin, hematin, and humic acid. Both kits showed the same performance on case work profiling andthe sensitivity of allele detection of P-6C was not significantly different from that of GF-IQC (p=0.144). The resultsof this study demonstrated that GF-IQC and P-6C with half volume reaction can produce good quality profiles andcould be applied for forensic DNA examination.

A case of "false tri-allelic pattern" on D7S820, caused by invasion of a short SE33 allele into the bins of D7S820.

Autosomal short tandem repeats (asSTR) serve as genetic markers for discriminating individuals and have been extensively used for criminal investigations as well as the establishment of genetic relationships. Tri-allelic pattern usually occurs due to chromosomal duplication, trisomy, and chimerism during mitotic division, but a false tri-allelic pattern at the D7S820 locus was encountered in our laboratory during the analysis of a case exhibit. DNA isolation from exhibit for profiling was done as per manufacturer's protocol. This is the first report which observed false tri-allelic pattern (10, 11, 14.1 allele) on D7S820 locus by analysis with GlobalFiler™ PCR Amplification Kit in Indian population. Findings were re-confirmed using other available asSTR kits in the laboratory, viz., AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit and PowerPlex® Fusion 6C System. Two alleles (10, 11) found at D7S820, apart from SE33 marker, showed homozygous condition, but one Off Marker (OMR) peak was observed before start of SE33 marker region with the analysis using PowerPlex® Fusion 6C System. As it has been confirmed that the OMR allele belongs to the SE33 locus, this could be possible because of the adjacent locations of the D7S820 and the SE33 in the GlobalFiler® PCR amplification kit. 14.1 allele appeared within the allelic window of D7S820. The false tri-allelic pattern was due to the overlapping of SE33 marker allele (1.2 repeat) with bin window of D7S820 Marker. This finding might create confusion for the establishment of genetic relationships. We, therefore, conclude that such uncommon observations with rare events should be carefully investigated and interpreted.

Evaluation of long-term storage effects on buccal cell DNA from untreated cards for STR profiling

The success of DNA profiling using long-term stored samples depends on the amount and quality of their recovered DNA templates. Physical and biochemical factors such as microbial activity, humidity and temperature contribute to DNA degradation. In this study, we used genomic DNA extracted from buccal cell samples that were stored for more than 4 years on Bode Buccal DNA Collector™ cards (Bode Technology, Virginia, USA) for typing 27 and 24 short tandem repeat (STR) loci using Powerplex® Fusion 6C system (Promega Corporation, Madison, USA) and Globalfiler™ Express kit (Thermo Fisher Scientific, USA), respectively. Our results demonstrated that the Buccal DNA Collector can be used as a collection medium for buccal swab samples that are not immediately analyzed or those that need to be retrospectively analyzed. There is a sign of DNA degradation which might well be expected because buccal cell samples were deposited on untreated filter paper and have been stored for a long period (> four years) at room temperature. However, STR allele calls were obtained from most of the buccal cell samples, especially when typed using the Powerplex® Fusion 6C system kit.

Determination of human identity from Anopheles stephensi mosquito blood meals using direct amplification and massively parallel sequencing

DNA obtained from biological evidence can link individuals to a crime scene. DNA is typically obtained from body fluids deposited on various substrates such as fabric or common household objects. However, other unusual sources of human biological material can also be used to generate DNA profiles. Here we show that short tandem repeat (STR) DNA profiles can also be obtained from single source and mixtures of human DNA in the blood meals of Anopheles stephensi mosquitoes. Using direct amplification with the PowerPlex® Fusion 6C System, we have determined that full and partial profiles can be obtained by assessing degradation of DNA at various times post-feed up to 20−24 h post-blood meal. Moreover, we can assign donor identity through both STR profiles, as well as through single nucleotide polymorphisms (SNPs) detected using massively parallel sequencing (MPS) with the Precision ID Identity Panel and Ion Chef™/Ion S5™ System up to 24−48 h post-blood meal. Based on the results from a total of 490 mosquitoes fed on 11 different sources of human blood, we conclude that both STR and SNP technologies can be applied to mosquito blood meals as effective forensic approaches to determine the identity of specific individuals and establish the timing of their presence at a crime scene.

Genomic diversity in the Goud population of Telangana, India inferred using twenty three autosomal marker PowerPlex® Fusion 6C System

Abstracts With the aim to investigate the genetic diversity of the Goud population of Telangana, India, 121 randomly selected healthy individuals were chosen for this study. The genetic diversity of the studied population was inferred using the PowerPlex® Fusion 6C system. A total of 257 alleles were observed in the studied population, along with the mean number of alleles 11.174 per locus. The locus SE33 showed the maximum number of observed allele i.e., 32, whereas locus TPOX showed the least number of observed allele i.e., 5. The studied population showed a wide range of allele frequencies ranged from 0.004 to 0.442. This wide range of allele frequency showed the diverse nature of the studied population, which is also supported by the broad range of observed Heterozygosity with the minimum value 0.628 to maximum 0.893, suggest the multidirectional gene flow in the studied population. The locus SE33 was also observed as the most polymorphic among all the studied loci. The combined value of the power of discrimination, power of exclusion, paternity index, and matching probability was found to be 1, 0.999999999178452, 9.86 × 108, and 4.5 × 10–28, respectively, for all the studied loci. Neighbor-Joining Tree and Principal Component Analysis (PCA) Plot showed the genetic affinity of the studied population with the Indian, central Indian, and populations of Uttar Pradesh and Jharkhand. This is the first global genetic report of genetic diversity in the Goud population of Telanagana using 23 markers PowerPlex® Fusion 6C system. The obtained genetic data of this study will be useful for forensic application, anthropological, and medical studies.

Copan microFLOQ® Direct Swab collection of bloodstains, saliva, and semen on cotton cloth

The microFLOQ® Direct Swab was tested by sampling diluted blood, semen, and saliva stains deposited on cotton cloth. DNA typing was performed using the PowerPlex® Fusion 6C System by direct PCR or a modified direct PCR. Direct PCR of swabs sampled the center of a stain, compared to their respective edge samplings, and had higher profile completeness and total relative fluorescent units (RFU) for all dilutions of blood and semen stains tested. The modified direct PCR used template DNA eluted from the swab head using the Casework Direct Kit, Custom and washes either contained 1-thioglycerol (TG) additive or no TG. Modified direct PCR had mixed results for blood, saliva, and semen stains, with semen stains showing significant differences in profile completeness (5% and 1%) and total RFU (neat, 5% and 1%) with the addition of TG to the Casework Direct Reagent. No significant difference was seen in any dilution of blood or saliva stains processed with the modified direct PCR, but profile completeness and total RFU were improved overall compared to stains swabbed with cotton swabs or 4N6FLOQSwabs™. This study supports the hypothesis that the microFLOQ® Direct Swab is able to collect minute amounts of DNA from cotton cloth and may be considered as an alternate pre-screening methodology in forensic biology casework.

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

PowerPlex® fusion 6C system: internal validation study

ABSTRACTThis work is aimed at describing the proceedings and parameters used to validate PowerPlex® Fusion 6C System, the polymerase chain reaction (PCR) amplification kit by Promega, for posterior implementation in the laboratorial routine of the Forensic Genetic Service. The PowerPlex® Fusion 6C System allows multiplex PCR, through simultaneous amplification and posterior detection by fluorescence of 27 loci. Characterization of the kit was made according to the laboratory's internal validation procedure based on validation guidelines from Scientific Working Group on DNA Analysis Methods. Some parameters were evaluated, such as specificity, analytical thresholds, sensitivity, precision, mixture studies, DNA control samples, a proficiency test and changes in the PCR-based procedures: final reaction volume and cycle number, changes in the reaction mixture for direct amplification. This kit proved to be very robust and the results are in concordance with previous developmental validation by the manufacturer. In some parameters, the results were better than expected.

Estimation of the number of contributors of theoretical mixture profiles based on allele counting: Does increasing the number of loci increase success rate of estimates?

DNA mixtures are more frequently encountered in casework due to increased kit sensitivity, protocols with increased cycle number, and requests for low copy number DNA samples to be tested. Generally, the first step in mixture interpretation is determining the number of contributors, with the most common approach of maximum allele count. Although there are previous studies regarding the accuracy of this approach, none have evaluated the accuracy with the newly expanded U.S. core STR loci. In this work, 4,976,355 theoretical mixture combinations were generated with the PowerPlex® Fusion 6C system which includes 23 autosomal STR loci and three Y-STR loci. The number of contributors could be correctly assumed for 100% two-person and 99.99% three-person mixtures, whereas, four-, five-, and six-person mixtures were correctly assumed in 89.7%, 57.3%, and 7.8% of mixtures, respectively. Y-STR analysis showed the 3 Y-STR markers are only accurate for two-person male mixtures (96.7%). This work demonstrates that maximum allele count using the expanded U.S. core loci is not much improved from previous smaller panels, reiterating that this method is not as accurate beyond three contributors.

Validation of the PowerPlex Fusion 6C system: a six-dye STR system for forensic case applications

The PowerPlex Fusion 6C system is a six-dye STR multiplex that allows co-amplification and fluorescent detection of 27 loci, including Amelogenin, 23 autosomal STR loci and 3 Y chromosome STR loci. In this study, this system was validated using upgraded 3130xl Genetic Analyzers for sensitivity, species specificity, stability, DNA mixtures, reproducability, case-type samples, stutter, concordance, and PCR conditions following the Scientific Working Group on DNA Analysis methods (SWGDAM) developmental validation guidelines and Chinese National Standard (GA/T815–2009). The results show this system is sensitive, species-specific, accurate and genotype-consistent compared with the PowerPlex ®21 system for detecting 100 samples, and robust for changes in several PCR conditions or exposure to PCR inhibitors. The results of casework indicate that Y-STR loci of the PowerPlex Fusion 6C system may help to recognize mixed and very-low-ratio male DNA. The population data of 274 unrelated Chinese Han individuals suggests that this multiplex system is suitable for human identification and paternity testing in Guangdong Han population. The data demonstrate that the PowerPlex Fusion 6C system is a robust and reliable STR-typing multiplex, and is especially advantageous for some case samples.

Genetic analysis of old skeletal remains from Korean War victims using PowerPlex® Fusion 6C and MiniSTR system for human identification

To analyze and identify the population of the Korean War victims by studying degraded old bones based genetic analysis, we examined 108 unrelated individual femurs except two low quality bones from total investigated 110 samples. In order to obtain significantly improved genetic profiles, amplification was performed using recently developed commercial PowerPlex® Fusion 6C system and miniSTR system. We have modified PCR condition to gain enhanced DNA profiling at a success rate. When each value of two different systems came out respectively as 92.35% and 91.05% only, it created 4.40% increased value which rated at 95.45% by using these two systems simultaneously. Especially, 100% showed at D1S1656, D2S441 and D12S391, which overlapped with miniSTR system. Total of 24 autosomal STRs had gene diversity values from 0.7751 to 0.9934; SE33, Penta E, and D2S1338 which were the best in that order. The most frequent value was 0.5170 at TPOX(8) in the frequencies ranging from 6 to 37 allele. Polymorphism information content, power of discrimination and exclusion, typical paternity index and heterozygote were the highest value at SE33, but TPOX appeared the lowest in all statistics. However the value of matching probability and homozygote showed the opposite result. Since above data anticipate that these outcomes confirm the similar patterns compared to previous reported Korean population papers, it verifies the population based genetic analysis using advanced these two systems is very helpful to increase an efficiency as well as a higher accuracy on identification of victims and also to check of identical samples.

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.

PowerPlex® Fusion 6C System: evaluation study for analysis of casework and database samples.

AimTo report on the successful analysis of amplicons obtained with PowerPlex® Fusion 6C System, a highly robust 27-plex genotyping kit developed for human identification laboratories, on the Applied Biosystems® 3500 Genetic Analyzer.MethodWe performed characterization and evaluation studies following the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines, examining several critical areas of kit performance. We report the results of sensitivity, robustness, heterozygous peak height ratio, precision, concordance, caseworks, and mixture interpretations. We tested sensitivity, using serial dilutions of control DNA.ResultsThe minimum amount of input DNA resulting in a full profile was 125 pg. Inhibition, inducted by urea, showed a progressively fragmentation of DNA and a full profile was obtained until 1M of inhibitor factor. To test the profile quality, casework samples were extracted with different extraction methods: Chelex®100, QIAmp DNA Micro Kit and Phenol-Chloroform extraction. The results demonstrated that extraction chemistries do not have affect on amplification performance. Concordance check was performed by typing some casework samples and comparing the typing results with those obtained with other available kits. Thus, concordance was expected and supported by the data.ConclusionReliable DNA typing results can be obtained using this new kit, demonstrating its effectiveness and utility in forensic analysis.

Developmental validation of the PowerPlex® Fusion 6C System

The PowerPlex® Fusion 6C System is a 27-locus, six-dye, multiplex that includes all markers in the expanded CODIS core loci and increases overlap with STR database standards throughout the world. Additionally, it contains two, rapidly mutating, Y-STRs and is capable of both casework and database workflows, including direct amplification. A multi-laboratory developmental validation study was performed on the PowerPlex® Fusion 6C System. Here, we report the results of that study which followed SWGDAM guidelines and includes data for: species specificity, sensitivity, stability, precision, reproducibility and repeatability, case-type samples, concordance, stutter, DNA mixtures, and PCR-based procedures. Where appropriate we report data from both extracted DNA samples and direct amplification samples from various substrates and collection devices. Samples from all studies were separated on both Applied Biosystems 3500 series and 6-dye capable 3130 series Genetic Analyzers and data is reported for each. Together, the data validate the design and demonstrate the performance of the PowerPlex® Fusion 6C System.