STR Kits Research Articles

Development of a qPCR assay for the quantification of canine autosomal DNA recovered from livestock attacks

The absence of a standardised method to quantify canine DNA recovered from livestock attacks leaves forensic providers without an important quality control step to help support their decision making. Typically used to normalise the amount of DNA for STR amplification, modern forensic DNA quantification approaches use qPCR of target genes and can also include an Internal Positive Controls (IPC) to determine the presence of PCR inhibitors. The co-amplification of livestock DNA alongside canine DNA has meant that previously developed qPCR methods are not suitable for use so a standardised approach is needed. This research describes the development of a Taq-man multiplex qPCR assay that simultaneously quantifies the autosomal MC1R and Y-specific SRY gene to determine the concentration of canine DNA recovered from attacked livestock. Data suggests that the method is robust and reproducible with no significant difference in the standard curves produced from multiple runs or from different DNA standards derived from different canines. Assay sensitivity of between 15 and 31 pg is consistent with other forensic quantification assays and also in line with the sensitivity of the two tested canine STR kits, Canine Genotype 2.1 Kit and CaDNAP Panels 1 and 2. The assay is highly specific to canines when tested against 163 different dogs representing 33 different breeds and no cross-amplification of non-target species’ DNA was observed even from livestock DNA tested at 31.25ng/µl. This strongly suggests that any DNA detected on evidence collected from attacked livestock is canine. The assay also shows robust tolerance to common livestock inhibitors continuing to amplify when inhibitor-spiked DNA samples were tested. Both mixed and inhibited DNA samples underwent STR typing using two canine forensic STR kits with data showing the Canine Genotype 2.1 Kit displaying pronounced cross-amplification of livestock DNA and and/or extensive PCR inhibition leading to the complete loss of amplification when using this kit. Conversely the CaDNAP Panels 1 and 2 showed little cross-amplification of livestock DNA and improved inhibitor tolerance suggesting that this approach was better suited for the analysis of livestock attack samples. Findings are discussed and the impact of the observations on future work in this area are explored.

Validation of the PowerPlex®35GY System: a novel eight-dye STR multiplex kit on the Spectrum Compact CE System.

The PowerPlex® 35GY System (Promega, USA) is an advanced eight-dye multiplex STR kit, incorporating twenty-three autosomal STR loci, eleven Y chromosome STR loci, one sex determining marker Amelogenin, and two quality indicators. This multiplex system includes 20 CODIS loci and up to 15 mini-STR loci with sizing values less than 250 bases. In this study, validation for PowerPlex® 35GY System was conducted following the guidelines of SWGDAM, encompassing sensitivity, precision, accuracy, concordance, species specificity, stutter, mixture, stability, and degraded DNA. The results from experiments demonstrated that the PowerPlex® 35GY System could effectively amplify DNA samples, with complete allele detection achieved at 125 pg. Moreover, over 90% of alleles from minor contributors were detected at a mixed ratio of 1:4. Additionally, the system was found to yield full profiles even in the presence of hematin, humic acid, and indigo. The PowerPlex® 35GY System demonstrated superior performance in the sensitivity and degraded DNA studies compared to a six-dye STR kit. Hence, it is evident that the PowerPlex® 35GY System is well-suited for forensic practice, whether in casework or for database samples. These findings provide strong support for the efficacy and reliability of the PowerPlex® 35GY System in forensic applications.

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.

A novel type of three band pattern at STR loci

SE33 or ACTBP2 is the most polymorphic locus in many national DNA databases and in the commercial STR kits used to type both crime scene samples and reference samples to populate these databases. We describe the molecular reason for a three band pattern of SE33 seen in several samples. A SNP in the flanking SE33 region causes the binding of the unlabelled D3S1358 primer. As a result, a “chimeric” PCR product of the labelled SE33 primer and the D3S1358 primer is generated that is smaller than the regular SE33 amplicon. We call this “Type 3 three band pattern” as the genetic base differs from the Type 1 three band pattern caused by somatic mosaicism and the Type 2 that results from copy number variation.

An inter-laboratory comparison of probabilistic genotyping parameters and evaluation of performance on DNA mixtures from different laboratories

Probabilistic genotyping (PG) is becoming the preferred standard for evidence interpretation, amongst forensic DNA laboratories, especially those in the United States. Various groups have expressed concern about reliability of PG systems, especially for mixtures beyond two contributors. Studies involving interlaboratory testing of known mixtures have been identified as ways to evaluate the reliability of PG systems. Reliability means different things in different contexts. However, it suffices here to think about it as a mixture of precision and accuracy. We might also consider whether a system is prone to producing misleading results – for example large likelihood ratios (LRs) when the POI is truly not a contributor, or small LRs when the POI is a truly a contributor. In this paper we show that the PG system STRmix™ is relatively unaffected by differences in parameter settings. That is, a DNA mixture that is analyzed in different laboratories using STRmix™ will result in different LRs, but less than 0.05% of these LRs would result in a different, or misleading conclusion as long as the LR is greater than 50. For the purposes of this study, we define LRs assigned using different parameters for the same mixtures as similar if the LR of the true POI is greater than the LRs generated for 99.9% of the general population. These findings are based on an interlaboratory study involving eight laboratories that provided twenty known DNA mixtures of two to four contributors and their individual laboratory STRmix™ parameters. The eight sets of laboratory parameters included differences in STR kits and PCR cycles as well as the peak, stutter, and locus specific amplification efficiency variances.

Improving the efficiency of Y-chromosome detection and the quality of STR typing in forensic casework with an in-house made qPCR and HRM system based on SYTO™ 9 chemistry

DNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and integrity of the DNA input in the PCR. In addition, the detection of male trace DNA provides key information for forensic investigation. AimTo evaluate the correlation between the quantification results obtained with the previously developed Amel-Y system, and its ability to detect Y-chromosome DNA by HRM, with the resulting STR profiles, and to ultimately show that Amel-Y can be routinely used in forensic casework to improve STR and Y-STR results. Material & methodsBiological samples derived from forensic casework (85 reference and 391 evidence samples) were quantified by the Amel-Y system (a duplex qPCR/HRM based on SYTO™ 9 chemistry) using Rotor-Gene 6000. STRs were amplified and analyzed with GeneAmp™ PCR System 9700 or Veriti™ Thermal Cyclers and ABI 3500 Genetic Analyzer, respectively. ResultsAfter DNA normalization, a total of 386 STR profiles were obtained (305 full and 81 partial). Sex typing by HRM was 100% successful in reference samples. Male DNA was detected by HRM in 210 evidence samples. 80/201 were mixed with an excess of female DNA. In addition, Amel-Y was able to detect Y-chromosome DNA in mixed samples that did not amplify the Y-variant of Amelogenin marker with commercial STR kits. The reproducibility and precision of the Amel-Y system were demonstrated (CVCt% ≤ 9.55) within the dynamic range analyzed (0.016–50 ng/µL; 41 independent runs). Amel-Y also proved to be compatible with other real-time PCR platforms. ConclusionWe demonstrated that Amel-Y is a robust quantification system that can be routinely used in forensic casework to obtain reliable autosomal STR profiles and can be suitable as a predictor for Y-STR typing success when male DNA is detected. HRM can be used as a rapid screening tool for male DNA detection in mixed samples. Alternative designs like Amel-Y offer independence from commercial quantification kits in forensic labs.

Genetic sexing of subadult skeletal remains

When subadult skeletons need to be identified, biological sex diagnosis is one of the first steps in the identification process. Sex assessment of subadults using morphological features is unreliable, and molecular genetic methods were applied in this study. Eighty-three ancient skeletons were used as models for poorly preserved DNA. Three sex-informative markers on the Y and X chromosome were used for sex identification: a qPCR test using the PowerQuant Y target included in PowerQuant System (Promega), the amelogenin test included in ESI 17 Fast STR kit (Promega), and a Y-STR amplification test using the PowerPlex Y-23 kit (Promega). Sex was successfully determined in all but five skeletons. Successful PowerQuant Y-target, Y-amelogenin, and Y-chromosomal STR amplifications proved the presence of male DNA in 35 skeletons, and in 43 subadults female sex was established. No match was found between the genetic profiles of subadult skeletons, and the elimination database and negative control samples produced no profiles, indicating no contamination issue. Our study shows that genetic sex identification is a very successful approach for biological sexing of subadult skeletons whose sex cannot be assessed by anthropological methods. The results of this study are applicable for badly preserved subadult skeletons from routine forensic casework.

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.

A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology.

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30-150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%-75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.

An analysis of data curated from 5 years of identifying human remains.

An archive of 5 years of cases involving the identification of human remains was curated, collecting information on: The sample type submitted, the number of STR loci yielding interpretable results, the kinship challenge posed, and the outcome for the case. A total of 129 cases of remains ID were investigated using manual DNA extraction and recovery methods with amplification of STR markers using the Power Plex 21 multiplex STR kit from Promega Corp. In 52 cases, blood spots collected by the ME were provided as sample and in 100% of those cases, probabilities of relatedness to the reference samples was ≥99%. In 77 cases, tissue other than blood was provided as a source of DNA. These other samples were grouped categorically into long bones (femur and tibia; 40 cases), skull bones/teeth (11 cases), other bones (16 cases), and tissue (normally adherent to bone) (10 cases). Reference samples provided for cases included alleged parents or child(ren) of the victim (86 cases), alleged full siblings of the victim (38 cases), or alleged second-order relatives (five cases). The overall success rate in confirming the identity of the source of the remains in these cases was 89.2%. Our results demonstrate that a laboratory can be often successful identifying human remains using methods easily implemented in any DNA typing laboratory.

Genetic research to evaluate plus stutter on D18S51 locus

The PCR amplification of STR loci typically produces a minor byproduct called minus stutter (or plus stutter) that is one repeat unit shorter (or one repeat unit longer) than the original product. These stutters cause some problems that affect the result of STR typing and complicate when comparing results of STR typing. It is necessary to remove the stutter in the forensic STR typing. Minus stutter is filtered out automatically by setting of the analysis software. On the other hand, plus stutter at most loci in STR Kits does not have such setting and it is in particular difficult to distinguish between the original peak and stutter peak.

Comparison of genotyping and weight of evidence results when applying different genotyping strategies on samples from a DNA transfer experiment.

In this study, we assessed to what extent data on the subject of TPPR (transfer, persistence, prevalence, recovery) that are obtained through an older STR typing kit can be used in an activity-level evaluation for a case profiled with a more modern STR kit. Newer kits generally hold more loci and may show higher sensitivity especially when reduced reaction volumes are used, and this could increase the evidential value at the source level. On the other hand, the increased genotyping information may invoke a higher number of contributors in the weight of evidence calculations, which could affect the evidential values as well. An activity scenario well explored in earlier studies [1,2] was redone using volunteers with known DNA profiles. DNA extracts were analyzed with three different approaches, namely using the optimal DNA input for (1) an older and (2) a newer STR typing system, and (3) using a standard, volume-based input combined with replicate PCR analysis with only the newer STR kit. The genotyping results were analyzed for various aspects such as percentage detected alleles and relative peak height contribution for background and the contributors known to be involved in the activity. Next, source-level LRs were calculated and the same trends were observed with regard to inclusionary and exclusionary LRs for persons who had or had not been in direct contact with the sampled areas. We subsequently assessed the impact on the outcome of the activity-level evaluation in an exemplary case by applying the assigned probabilities to a Bayesian network. We infer that data from different STR kits can be combined in the activity-level evaluations.

Characterizing the amplification of STR markers in multiplex polymerase chain displacement reaction using massively parallel sequencing

Polymerase chain displacement reaction (PCDR) showed advantages in forensic low-template DNA analysis with improved amplification efficiency, higher allele detection capacity, and lower stutter artifact than PCR. However, characteristics of STR markers after PCDR amplification remain unclarified for the limited resolving power of capillary electrophoresis (CE). This issue can be addressed by massively parallel sequencing (MPS) technology with higher throughput and discriminability. Here, we developed a multiplex PCDR system including 24 STRs and amelogenin. In addition, a PCR reference was established for comparison. After amplification, products were subjected to PCR-free library construction and sequenced on the Illumina NovaSeq system. We implemented a sequence-matching pipeline to separate different amplicon types of PCDR products from the combination of primers. In the sensitivity test, the PCDR multiplex obtained full STR profiles with as low as 125 pg 2800M control DNA. Based on that, single-source DNA samples were tested. First, highly concordant genotypes were observed among the PCDR multiplex, the PCR reference, and CE-based STR kits. Next, read counts of different PCDR amplicon types were investigated, showing a relative abundance of 78:12:12:1 for the shortest amplicon S, the two medium amplicons M1 and M2, and the longest amplicon L. We also analyzed the stutter artifacts for distinct amplicon types, and the results revealed the reduction of N − 1 and N − 2 contraction stutters, and the increase of N + 1 and N + 2 elongation stutters in PCDR samples. Moreover, we confirmed the feasibility of PCDR for amplifying degraded DNA samples and unbalanced DNA mixtures. Compared to the previous proof of principle study, our work took a further step to characterize the complete profile of STR markers in the PCDR context. Our results suggested that the PCDR-MPS workflow is an effective approach for forensic STR analysis. Corresponding findings in this study may help the development of PCDR-based assays and probabilistic methods in future studies.

Detection and analysis of DNA mixtures with the MiSeq FGx®

Recognizing and interpreting mixtures are challenges that occur frequently in forensic casework. Therefore, any new analysis methods that are implemented must handle the challenges of mixed forensic samples. Next generation sequencing offers advantages over capillary electrophoresis in amplicon multiplexing and degraded sample analysis; however, advantages with mixed samples rely heavily on the advancement of user-friendly analysis software. This research analyzed samples with the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx® and compared them with the GlobalFiler™ STR Kit for capillary electrophoresis. Metrics tested for both chemistries included concordance, limits of detection, and mixture analysis. Data analysis for mixture samples was completed with the MixtureAce™ plug-in and ArmedXpert™ software. Next generation sequencing offered distinct advantages in limits of detection and isoallele heterozygosity but suffered from increased variability in stutter and allele count ratios compared to capillary electrophoresis.

Comparison between various DNA sterilization procedures applied in forensic analysis

BackgroundThe advanced sensitive STR kits applied in forensic DNA typing techniques can cause challenging issues when evidence samples are contaminated with minute quantities of DNA from another source such as forensic analysts or crime scene examiners.ResultsIn this study, laboratory air and surfaces, gloves, tools, and equipment were evaluated as potential sources of contaminating DNA. Different sterilization methods were tested for their ability to efficiently eliminate DNA in a sample. Inactivation methods included 10% bleach, ethanol, UV light, and DNA-ExitusPlus IF. Exposure to the different inactivation protocols for varying periods of time was performed in two lab settings: low template DNA and DNA database labs. Surfaces were swabbed and any adhering DNA was quantified using HID real-time PCR. Results were detected using HID Real-Time PCR Analysis Software v1.2 and GeneMapper ID-X Software v1.4.ConclusionsIt was concluded that most of the DNA decontamination methods are not suitable for highly sensitive and precision STR kits such as GlobalFiler PCR Amplification Kit. The most suitable tested method was using DNA-ExitusPlus IF with the incubation time increased from 10 to 15 min.

A single direct amplification method for forensic casework references on a variety of substrates

A single direct amplification method for buccal and blood reference samples, on different substrates, containing a wide range of cellular material, was developed. This method uses the GlobalFiler™ STR kit, which was designed for amplification of previously purified and quantified DNA. Buccal samples on cotton swabs, Bode collectors, and Whatman® FTA® cards, and liquid blood, blood on Whatman® 903 and FTA® cards, and autopsy bloods were examined. A first pass success rate, that is, complete concordant profiles with no GeneMapper™ ID-X quality flags, of > 95% was obtained. This method involves the lysis of reference samples (tips of cotton swabs, double 4.8 mm diameter Bode or FTA® buccal punches, and single 4.8 mm diameter blood punches on Whatman® 903 or FTA® cards) in 100 µl of Prep-n-Go™ lysis buffer, and amplification of 2 µl of the lysate with 25 cycles. Data collection is optimized by use of the spectral pull-up reduction and off-scale recovery features of the AB3500 Genetic Analyzer Data Collection version 4 software, and by injection of PCR products for two different lengths of time (15 and 7 s). Data was analyzed with GeneMapper™ ID-X version 1.6, which allowed an increased signal dynamic range (maximum peak height of 64000 RFU), and by employing a 10% global filter, and a 20% global stutter filter. Complete GlobalFiler™ profiles were generated from as little as 5 µl of blood, 1 buccal swipe on cotton swab and Bode collectors, and collection of buccal cells deposited on the foam heads of EasiCollect™ FTA® collectors for 5 s.

Total allele count distribution (TAC curves) improves number of contributor estimation for complex DNA mixtures

As the forensic community is transitioning to probabilistic genotyping and the use of likelihood ratios to assign probative weight to DNA mixtures, the assessment of the number of contributors (NOC) needs to be more robust for mixture interpretation. However, NOC assessment can be challenging for low-template and/or high order mixtures. Here, we present a quick and easy-to-use tool to help with NOC estimation: total allele count curves (TAC curves). TAC curves for two to seven contributors were generated using sets of 20,000 in silico mixtures, for five populations (African American, Caucasian, Asian, Apache and Native Alaska) and for commonly used commercial STR kits (GlobalFilerTM, PowerPlex® Fusion, PowerPlex® ESX 17 and IdentifilerTM). To assess the performance of TAC curves, the NOC was evaluated for 80 mixtures, with and without use of the curves. Results show that TAC curves allow for a better NOC assessment as correct evaluations rose from 10% when using maximal allele count (MAC) to 65% when also using TAC for four to six contributor mixtures. Supplemental data for this article is available online at http://dx.doi.org/10.1080/00085030.2022.2028359 .

Application of ITO Method in Determination of Special Half-Sibling Relationship.

To identify whether the relationship between Zhang A, Zhang B, Zhang C and Zhang X is the half-sibling relationship whose mother is sister (hereinafter referred to as the special half-sibling relationship) or the common first cousin relationship and discuss the application of ITO method in discriminating the special kinship. DNA was extracted from blood stain of four identified individuals, PowerPlex® 21 System and AGCU 21+1 STR kit were used to detect autosomal STR genetic markers. Investigator® Argus X-12 QS kit was used to detect the X chromosome STR genetic markers, the special half-sibling index (SHSI) and first cousin index (FCI) and their likelihood ratio (LR) were calculated by ITO method. The LR results of SHSI to FCI, which were calculated based on autosomal STR genotyping and the analysis of X-STR genotyping results suggested that the relationship between Zhang A, Zhang B, Zhang C and Zhang X was inclined to be a special half-sibling relationship. For the identification of special kinship, it is necessary to comprehensively apply various genetic markers according to the case. After the conclusion that shared alleles cannot be excluded from the analysis, ITO method can be further used to establish discriminant assumptions according to the specific case to obtain objective and reliable identification opinions.

Evaluation of diallelic STR markers with inter-population allelic database for their usefulness in paternity trios in the central Indian population

Background Most of the forensic DNA laboratories have migrated to new generation STR kits of 6 dye chemistry with more than twenty autosomal STRs. The population-specific database of such STR markers is lacking in many regions. Aim To evaluate the effect of the inter-population database in 100 paternity trios with no inconsistencies at 23 STRs. Subjects and methods 100 paternity trios were evaluated considering inter-population allelic frequency values for calculation of Combined Paternity Index (CPI) and Probability of Paternity (POP). Results No significant variation (p < 0.05) among the allele frequencies at the interpopulation level was observed. The number of obligate alleles and the likelihood of transferring obligate alleles from the putative father showed a positive correlation (p < 0.005) with Power of Discrimination (PD), Polymorphic Information Content (PIC), Power of Exclusion (PE), Paternity Index (PI), Observed and Expected Heterozygosity (Ho and He), and a statistically significant negative correlation (p < 0.005) with Matching Probability (Pm). The average Combined Paternity Index (CPI) and Probability of Paternity (POP) did not show any statistically significant difference (p < 0.05) at the interpopulation level. Conclusion The allelic database showed no effect on the CPI and POP in 100 paternity trios. This suggests no urgent need for using population-specific databases for statistical evaluation of paternity trios without inconsistencies.

Comparison of six commercially available STR kits for their application to touch DNA using direct PCR

With an increase in the application of direct PCR to items of forensic relevance, as well as the array of STR kits available for amplification, the need for a comprehensive investigation into the optimum STR panel for this workflow has arisen. Here we examine the relative STR amplification success of touch DNA on a range of substrates, with surface properties typical of those found in forensic investigation, using six commercially available STR kits: GlobalFiler®, Identifiler® Plus, Identifiler® Direct, VeriFiler™ Plus, Investigator® 24Plex QS, and PowerPlex® 21. We report on the percentage of possible donor alleles amplified per profile, and the number of samples that resulted in informative genetic data (≥12 autosomal alleles). We also include comment on the ease of interpretation of the resulting electropherograms for each of these six STR kits, when applied within a direct PCR workflow. Donors of known shedder status deposited DNA by handling one of five substrates (glass slide, matchstick, insulated wire, circuit board, and a plastic ziplock bag) for 15 s, 15 min post-handwashing with water. Each item was touched in triplicate by each volunteer for amplification with each STR kit resulting in a dataset of 720 samples. The difference in the number of profiles considered to be informative was found to be statistically significant when comparing STR kits (p = 0.0011) and donors (p = 2e-07), but not when considering substrates (p = 0.15). Identifiler® Plus amplification resulted in the highest profile coverage and second highest percentage of informative profiles. VeriFiler™ Plus generated informative profiles in the largest number of samples (94%) with PowerPlex® 21 amplification resulting in the fewest (79%). There was no significant difference between Investigator® 24Plex QS and GlobalFiler® in any empirical consideration; however, baseline noise and artefact presence made Investigator 24PlexQS profiles more difficult to analyse.