STR Multiplex System Research Articles

Development and Validation of a Novel 29-plex STR Multiplex System for Legal and Forensic Science

Development and Validation of a Novel 29-plex STR Multiplex System for Legal and Forensic Science

Development and validation of a forensic six-dye multiplex assay with 29 STR loci.

This paper describes the development and validation of a novel 31-locus, six-dye STR multiplex system, which is designed to meet the needs of the rapidly growing Chinese forensic database. This new assay combines 20 extended-CODIS core loci (D3S1358, D5S818, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D8S1179, D18S51, D16S539, D13S317, FGA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045), nine highly polymorphic loci in Chinese Han population (D3S3045, D6S1043, D6S477, D8S1132, D10S1435, D15S659, D19S253, Penta D, and Penta E), and two gender determining markers, amelogenin and Y-Indel, which could amplify DNA from extracts, as well as direct amplification from substrates. To demonstrate the suitability for forensic applications, this system was validated by precision and accuracy evaluation, concordance tests, case sample tests, sensitivity, species specificity, stability, stutter calculation, and DNA mixtures, according to the guidelines described by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and regulations published by the China Ministry of Public Security. The validation results indicate the robustness and reliability of this new system, and it could be a potentially helpful tool for human identification and paternity testing in the Chinese population, as well as facilitating global forensic DNA data sharing.

Evaluation of two 13-loci STR multiplex system regarding identification and origin discrimination of Brazilian Cannabis sativa samples.

According to the Brazilian Federal Police (BFP), the Brazilian Cannabis sativa illicit market is mainly supplied by drugs originated from Paraguay and Northeastern Brazil (Marijuana Polygon region). These two known routes, the increasing indoor cultivations (supported by online market), and drugs from Uruguay are also in BFP's sight. Forensic tools to aid police intelligence were published in the past years. In genetics, microsatellites have gained attention due to their individualization capability. This study aims to evaluate the effectiveness and efficiency of two STR multiplex systems previously proposed in 94 Cannabis sativa samples seized in Brazil. Principal coordinate analyses (PCoA), forensic parameters, and genetic structure analysis were executed. Both panels were effective in individualizing and origin discriminating all samples, and the system proposed in 2015 demonstrated better results. For this marker set, the probability of identity for a random individual is approximately one in 65 billion; also, the PCoA shows a clear genetic distinction among samples according to its origin. Bayesian inference populational structure analysis indicated a significant genetic diversity among seizure groups, matching with its origin. Overall, the STR multiplex systems were able to achieve its purpose in individualizing and differentiating, according to geographic region, Brazilian Cannabis sp. samples.

Forensic effectiveness and genetic distribution of 23 autosomal STRs included in Verifiler PlusTM multiplex in a population sample from Madhya Pradesh, India.

We report here the first ever global study on genetic polymorphism using a Verifiler PlusTM autosomal STR multiplex system. The study evaluated genetic characteristics of 23 autosomal STRs in 200 unrelated residents of Guna district of Madhya Pradesh, India. Allele frequencies and forensic parameters are reported. Population comparison analysis was also performed using NJ tree and PCA plot. Penta E marker showed highest power of discrimination (0.938) among all 23 studied markers. The study also presents the first ever global forensic assessment in Indian population on D6S1043 marker (PD 0.937). The results demonstrated that all the 23 markers were highly polymorphic and the Verifiler PlusTM kit is suitable for forensic purposes in Indian population.

Comparison of minidogfiler and "ASCH" STR multiplex systems for preliminary estimation of variability within wolf´s like dog breeds

This study was undertaken to estimate basic population parameters and compare resolution capabilities of two chosen STR panels (Mini-DogFiler, MDF; “ASCH”). Wolf´s like dog breeds, namely Czechoslovakian wolfdog (n = 55), Saarloos wolfdog (n = 22), and samples of the Eurasian wolf (n = 21) were selected for their close genetic relationship. Also, German Shepherd (n = 26) breed was included as a donor of dog genome for the Czechoslovakian wolfdog.Overall 228 alleles were detected using 20 STR markers (MDF 11 and “ASCH” 9 loci). Number of alleles ranged from 3 to 23 per locus, with median 11 alleles per locus for MDF and 10 for “ASCH”. Both panels showed similar level of variability in the means of number of alleles per locus (k) and observed heterozygostity (HO) as there were not found statistically significant differences (Mann-Whithey U-test; p = 0.85 for k, p = 0.88 for HO). A group of wolves showed the highest variability unlike Saarloos wolfdog with very low variability within all measured parameters, although such differences were statistically significant only for k parameter of MDF panel (Kruskal-Wallis ANOVA; p = 0.0003) and no other groups were different. Both microsatellite panels were able correctly distinguish 3 dog breeds and wolfs as was verified by STRUCTURE programme and several potential hybrids were identified. Moreover, this pilot analysis amenable creation of allelic ladders for MDF and “ASCH” panels and thus ensure consistency and reliability of future study with extended number of samples.

Application of 13 loci STR multiplex for cannabis sativa genotyping

Our work describes an application of a STR multiplex systems for 13 loci, previously developed and validated in our laboratory following international guidelines, on 4 cannabis specimens seized by Anti-Narcotic Italian Police.STR loci were chosen from the set of publicly available STRs for cannabis sativa and among the highly polymorphic available in literature.Gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC–MS) analysis revealed and confirmed four comparable molecular spectrums, approximatively descriptive of the same plant/seizure origin. Due to these uncertain results, investigative authorities approved the genetic analysis. DNA profiling allowed to identify the same genetic profile and consequently the same origin and individualization of the specimens analysed.

Forensic genetic analysis of the population of Gujarat with PowerPlex 21 multiplex system

The present investigation assessed the allele frequencies and forensic parameters for the loci included in PowerPlex 21 multiplex system (Promega, USA) in 101 unrelated individuals residing in the state of Gujarat, India. The study likewise introduces the first global report on polymorphism on Penta D and Penta E autosomal STR loci from the population of Gujarat, India. The obtained outcomes in the study revealed that the considered STR multiplex system is highly polymorphic and suitable for forensic genetic analysis.

Comparison of different STR Typing for a Special Allele at D7S820 Locus by Using Ten Different STR Multiplex System

During a DNA database construction and population study in China we observed a special genotype at D S locus which was misjudged by seven out of ten different STR multiplex systems Allele typing results acquired by using ten diverse STR multiplex kits PowerPlex reg Fushion C nbsp PowerPlex reg nbsp PowerPlex reg nbsp ACGU reg EX nbsp AppliedBio reg HuaXiaBaijin nbsp SureID reg PanGlobal nbsp GoldeneyeTM A nbsp GoldenEyeTM nbsp A nbsp AppliedBio reg GlobalFiler HuaDa reg YanHuagn and then analyzed by GeneMapper reg software In this paper we conclude the reasons for the misjudgment of this allele as well as how to reduce these kinds of errors during STR analysis

Comparison of different STR typing for a special allele at D7S820 locus by using ten different STR multiplex system

During a DNA database construction and population study in China we observed a special genotype at D S locus which was misjudged by seven out of ten different STR multiplex systems Allele typing results acquired by using ten diverse STR multiplex kits PowerPlex reg Fushion C nbsp PowerPlex reg nbsp PowerPlex reg nbsp ACGU reg EX nbsp AppliedBio reg HuaXiaBaijin nbsp SureID reg PanGlobal nbsp GoldeneyeTM A nbsp GoldenEyeTM nbsp A nbsp AppliedBio reg GlobalFiler HuaDa reg YanHuagn and then analyzed by GeneMapper reg software In this paper we conclude the reasons for the misjudgment of this allele as well as how to reduce these kinds of errors during STR analysis

Forensic genetic analysis of population of Madhya Pradesh with PowerPlex Fusion 6C\u2122 Multiplex System

Performance of PowerPlex Fusion 6C kit (PP F6C) was assessed in 374 unrelated individuals belonging to Madhya Pradesh, an Indian state. The study evaluated the forensic parameters for the loci included in PP F6C Multiplex System. The combined discrimination power (CPD) and combined exclusion power (CPE) were 1 and 0.999999995, respectively, for all 23 autosomal STR loci. SE33 showed the greatest power of discrimination (0.990) in the studied population, whereas TPOX showed the lowest (0.843). The availability of three Y-STR loci in the Multiplex System is suitable for assessing male contribution and amelogenin deletion in a single Multiplex PCR simultaneously. The study also presents the first global report on polymorphism in the Indian population on SE 33 autosomal STR loci and PP Fusion 6C Multiplex System. The results revealed that the studied STR Multiplex System is highly polymorphic and suitable for forensic purposes.

Genetic data for PowerPlex 21™ autosomal and PowerPlex 23 Y-STR™ loci from population of the state of Uttar Pradesh, India.

In the present study, the statistical forensic parameters were evaluated for the loci present in PowerPlex 21 autosomal and PowerPlex 23 Y-STR multiplex systems in 168 unrelated individuals living in the state of Uttar Pradesh, India. The combined discrimination power (CPD) and combined exclusion power (CPE) was 1 and 0.999999 respectively for all 20 autosomal STR loci. Penta E showed the greatest (0.980) and CSF1PO showed the lowest (0.855) power of discrimination in the studied population. The haplotype diversity for 23 Y-STR loci was observed to be 0.999. The study also presents the first global report on polymorphism on D1S1656, D6S1043 and D12S391 autosomal STR loci in the Indian population. The resulting data revealed that these STR multiplex systems are highly polymorphic and can be used for forensic purposes.

Validation and implementation of the Investigator® 24plex QS kit for forensic casework

Advanced commercial STR multiplex systems containing the expanded CODIS core loci have been developed to achieve robust results for a wide range of inhibited and degraded forensic samples. To address these requirements and challenges, the new Investigator® 24plex QS kit (QIAGEN) was evaluated in this study which coamplifies all 23 recommended markers using 6-dye technology and incorporates an innovative internal control, the Quality Sensor (QS). The Quality Sensor, containing resultant alleles “Q” and “S”, which is unique and patent pending, allows differentiation between robust amplification, degradation, inhibition, lack of amplification of DNA and failed PCR amplification. The performance of the Investigator® 24plex QS kit was evaluated using various sample types, a total of 130 samples from 25 forensic caseworks commonly encountered in our forensic laboratory during law enforcement investigation (homicide, sexual assault, robberies, criminal possession of a firearm, possession of a controlled substance, and property crimes-burglaries). The data indicates that the Investigator® 24plex QS kit produces an excellent “First Pass Success Rate” (full profile obtained with one amplification and one CE injection) with conclusive and reliable profiles, even when amplifying degraded or inhibited samples. Based on the results of this study, Investigator® 24plex QS Kit is rapid, achieves optimal results with difficult casework evidence samples and increases the detection of a minor contributor in mixtures. As an additional benefit, it improves the laboratory workflow due to the additional information given by the “Quality Sensor” and assists forensic experts in the selection of the most appropriate rework strategy.

Genetic polymorphisms of 22 autosomal STR loci in Chinese Han population

Microreader™ 23sp ID system was a new STR multiplex system developed for forensic application, but there was a lack of population data in the Chinese Han population. This kit contained 21 non-CODIS STR loci (D6S477, D18S535, D19S253, D15S659, D11S2368, D20S470, D1S1656, D22-GATA198B05, D7S3048, D8S1132, D4S2366, D21S1270, D13S325, D9S925, D3S3045, D14S608, D10S1435, D12S391, D2S1338, D17S1290, D5S2500), one CODIS STR locus (D16S539) and the amelogenin locus. In this study, a population sample in Chengdu Han population was analyzed with the kit. Forensic genetic parameters and probability values of the Hardy–Weinberg equilibrium (HWE) were calculated with software PowerStats v1.2 and Arlequin v3.5. Allele frequencies of the 22 STR loci and further forensic genetic parameters were obtained. The results suggested that Microreader™ 23sp ID system can provide informative polymorphic data for identification and parentage testing in Chinese Han population.

Developmental validation of the AGCU 21+1 STR kit: a novel multiplex assay for forensic application.

In this study, we describe the developmental validation assay performed on a novel designed STR multiplex system, AGCU 21+1 STR kit. This kit contains a sex-determining locus amelogenin and 21 noncombined DNA index system STR loci, that are, D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D1S1627, D3S4529, D2S441, D6S1017, D4S2408, D19S433, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D9S1122, D2S1776, D10S1435, and D5S2500. The 21+1 kit was validated by a series of tests including optimized PCR conditions, sensitivity, precision and accuracy, stutter ratio, DNA mixture, inhibitors, and species specificity according to the revised validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Our results in this study show that the kit is a useful tool for forensic application.

High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6–200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.

A new strategy for sperm isolation and STR typing from multi-donor sperm mixtures.

Mixed semen stains from multiple contributors are challenging samples in sexual assault casework, and it is crucial to obtain the DNA profiles of different donors to allow the evidence to play an important role in investigations and judicial proceedings. Current standard procedures, including preferential lysis, are incapable of separating single-source sperm from multiple male donors. Mixed profiles are often obtained and may not directly exclude or identify suspects. In this case, computational methods for mixture interpretation are often used, which rely on different types of calculation models. Here, we explored a new strategy for sperm cell isolation and detection from mixtures. It is a direct way to obtain genotypes of different sperm donors compared to computation-based mixture interpretation. Laser capture microdissection (LCM) and low volume-PCR (LV-PCR) were used for single sperm isolation and detection. The platform was sensitive; profiling of a single sperm cell generated a minimum of 13-16 loci in 73.1% of Y short tandem repeat (Y-STR) assays. A new Y-STR and autosomal STR multiplex system (YA-STR) were optimized by the combination of the Y-STR locus and 10 autosomal STR (auto STR) loci. The Y-STR locus acted as a tag to discriminate profile groups from different donors. Subsequently, consensus auto STR profiles of various persons could be received. The accuracy and availability of this method were evaluated on a three-donor semen mixture and found to be effective for the resolution of a multi-donor sperm mixture.

I-DNASE21 system: Development and SWGDAM validation of a new STR 21-plex reaction

I-DNASE21 is a new STR multiplex system that amplifies 21 STR autosomal loci, plus the amelogenin locus in one reaction. This system has been designed to analyze all the STR loci included in the Combined DNA Index System (CODIS), Interpol Standard Set of Loci (ISSL), Extended European Standard Set (ESS-Extended), UK National Criminal Intelligence DNA Database (NDNAD) and German Core loci (GCL). This manuscript presents the validation of the I-DNASE21 system according to the revised guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM). The results of this validation, added to the extremely high discriminatory power showed, suggest that I-DNASE21 could be a potentially helpful tool for identification and kinship determination even in complex paternity cases.

Consideration of the probative value of single donor 15-plex STR profiles in UK populations and its presentation in UK courts

The adoption of new 15 locus STR multiplex systems into UK forensic science would be facilitated by agreed guidelines for reporting the strength of DNA evidence using likelihood ratios. To facilitate such an agreement, we present an analysis of previously published UK allele frequencies for white Caucasian, Afro-Caribbean and Indo-Pakistani populations and investigate their effect on likelihood ratios for single donor profiles. We consider the implication of the five additional loci and suggest a procedure for reporting likelihood ratios for 15-plex STR profiles.

Consideration of the probative value of single donor 15-plex STR profiles in UK populations and its presentation in UK courts II

The adoption of new 15 locus STR multiplex systems into UK forensic science would be facilitated by agreed guidelines for reporting the strength of DNA evidence using likelihood ratios. To facilitate such an agreement, we present an analysis of previously published UK allele frequencies for white Caucasian, Afro-Caribbean and Indo-Pakistani populations and investigate their effect on likelihood ratios for single donor profiles. We consider the implication of the five additional loci and suggest a procedure for reporting likelihood ratios for 15-plex STR profiles.

Allele Frequency Distribution for 9 STR Loci in the Tamil Population of Malaysia

DNA database was obtained from unrelated random Tamil individuals in Malaysia. The DNA was extracted by the salting out procedure (1). Ten ng target DNA was co-amplified using the commercial typing kits, Promega GeneprintTM STR multiplex (CTT, FFv and STR III) systems, according to the manufacturer’s instructions. Assignment of alleles was made by visual comparison between the commercially supplied reference allelic ladders and the amplified samples at the corresponding locus. Data were analyzed as per the methods already reported (2–4). No deviations from equilibrium were observed. The power of discrimination ranges from 0.8458 to 0.9485. The complete dataset can be accessed at www.ppsk.ugm.mg