Genetic variation in a Japanese population,using the multiplex 24 STRs analysis system

Allele frequency distributions for 24 short tandem repeat (STR) loci were determined using the PowerPlex^R Fusion System (Promega) in 407 Japanese samples. The most informative locus among the 22 STR loci, excluding Amelogenin and DYS391, was Penta E (power of discrimination (PD) = 0.98), while the least informative was TPOX(PD = 0.831). The 22 loci combined matching probability (MP) was calculated to be 4.13 × 10⁻²⁶. These parameters indicated the usefulness of this 24 STR analysis in forensic personal identification and parentage testing among Japanese population.     

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits

Abstract: Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpF?STR^® MiniFiler? kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpF?STR^® MiniFiler? and Identifiler? kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler? kit, 75% of the same loci profiles were determined by MiniFiler? kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler? kit could be a useful complement to conventional STR kit analysis of ancient samples.     

D5S818 Typing Discrepancy Between PowerPlex® Fusion and Other STR Kits Including GlobalFiler® Caused by a One‐base Deletion in 31 Nucleotides Upstream of the Repeat Region

Short tandem repeat (STR) typing is widely used in forensic investigation. When the same DNA sample is analyzed with different STR typing kits, a typing discrepancy is occasionally observed. In this study, we examined the cause of a typing discrepancy in a sample at D5S818 locus. This sample was designated as 10, 12 using Identifiler^®, Identifiler^® Plus, GlobalFiler^®, PowerPlex^® 16HS, and PowerPlex^® 18D, but as 9.3, 12 using PowerPlex^® Fusion. Sequencing results indicated that the shorter allele in the sample had a deletion (U31Tdel) at 31 nucleotides upstream of the repeat region (AGAT)₁₀. This deletion was located in the binding site of the published D5S818 forward primer in PowerPlex^® 16 and was only 9 and 11 nucleotides downstream of our estimated 5′ end position of D5S818 forward primer in GlobalFiler^® and PowerPlex^® 18D, respectively. We also examined the effect of primer length on the heterozygous peak balance in this sample.     

Simplified Direct PCR Method for Reference Buccal Samples Using a Non-FTA Card by Omitting the Pretreatment Step

When using non-FTA cards in commercial multiplex STR kits for direct PCR, pretreatment steps with specific buffers are recommended. Here, we designed a rapid direct PCR method utilizing a non-FTA card, Oral Cell Sampling Kit, by omitting the pretreatment step involving Prep-n-Go™ Buffer, and it showed compatibility with the GlobalFiler™ Express PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit, and PowerPlex^® Fusion system. To optimize the PCR conditions, we tested the method with different final PCR volumes and cycles. Finally, we conducted a performance test using 50 Korean buccal samples and confirmed the high performance of the method, detecting more than 90% of the samples with full profiles when using GlobalFiler™ PCR Amplification Kit and PowerPlex^® Fusion system at 29 cycles in a 10 μL final PCR volume. Thus, we report a simple direct PCR set-up to analyze reference samples collected using a non-FTA card manufactured in Korea.     

An Investigation of PCR Inhibition Using Plexor®‐Based Quantitative PCR and Short Tandem Repeat Amplification

A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor^® real‐time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and C_T takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex^® 16 HS System. The overall results demonstrate that real‐time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.     

Getting Ahead: Extraction of DNA from Skeletonized Cranial Material and Teeth

Between 1990 and 2018, the Defense POW/MIA Accounting Agency submitted 2177 cranial elements and 1565 teeth to the Armed Forces Medical Examiner System—Armed Forces DNA Identification Laboratory for DNA testing. In an effort to identify missing United States service members, materials were recovered from wartime losses inclusive of World War II, the Korean War, and Southeast Asia. Using four different DNA extraction protocols, DNA testing was performed using mitochondrial DNA Sanger sequencing, modified AmpFlSTR^® Yfiler?, AmpFlSTR^® MiniFiler?, PowerPlex^® Fusion, or Next Generation Sequencing. This paper aims to provide optimal strategies for the DNA testing of skeletonized cranial materials. Cranial elements produced the most consistent results in Sanger sequencing using an organic purification; however, teeth were most successful for the same platform with an inorganic purification. The inverse is true for STR testing of cranial bones. Of the cranial elements, the temporal provided the most consistent results.     

Powerplex® Y 23 System: Molecular characterization of a null allele at locus DYS549

We observed a null allele pattern at locus DYS549 in a male subject from North-East Italy typed with the PowerPlex^® Y 23 System (Promega). To investigate whether this pattern was due to the presence of a microdeletion/mutation in primer binding sites or in the locus target region, the sample was amplified with our designed DYS549 primers obtained from GenBank sequence (GDB: 515022). After amplification, a normal hemizygous genotype at this locus was generated, thus indicating the presence of a point mutation in the binding site of the original primer set of PowerPlex^® Y 23 System (Promega). This was further confirmed by sequence analysis, carried out with the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), according to the manufacturer's instructions. Sequences were run on the ABI Prism 3130 Genetic Analyzer (Applied Biosystems) and analyzed using the Sequencing Analysis v.5.3.1 and the SeqScape v2.6 softwares (Applied Biosystems). Ascertainment of the frequency of null alleles generated from variations at primer binding sites of short tandem repeats loci is of great importance in forensic genetics.     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

The effect of cleaning agents on the ability to obtain DNA profiles using the Identifiler™ and PowerPlex® Y multiplex kits

Abstract: A year after the introduction of Identifiler? into the forensic DNA laboratories of the Institute of Environmental Science and Research Limited (ESR), increasing occurrences of dropout of the three loci, D7S820, D18S51, and FGA, were observed in samples where the DNA was not degraded and sufficient DNA was present that full DNA profiles were to be expected. The dropout was either partial or complete at these loci. Full profiles could sometimes be obtained by reamplification of samples using the same input amount of DNA. After a thorough investigation of the methods and procedures used in the laboratory, the cause of this inhibition was identified as the cleaning agent TriGene? ADVANCE. This was determined after the deliberate addition of varying amounts of different cleaning reagents into the DNA amplification reactions. At concentrations of 0.004% TriGene? ADVANCE caused inhibition resulting in tri‐loci dropout. At concentrations of 0.04% and higher, complete inhibition was observed. An effect was also seen on the amplification of samples using the Y STR profiling system PowerPlex^®Y. This work highlights the importance of checking all reagents and chemicals prior to use, even those with no apparent direct influence on the DNA profiling process.     

Genetic polymorphism of 17 STR loci for forensic use in Chinese population from Shanghai in East China

Allele frequencies for 17 STR loci found in Identifier kit and PowerPlex^®16 Monoplex System were determined in a sample of 1000 unrelated individuals living in Shanghai in East China. The values of observed heterozygosity (Ho), power of discrimination (PD), probability of paternity exclusion (PE) and polymorphism information content (PIC) were calculated. All loci were in accordance with Hardy–Weinberg equilibrium (p < 0.05). The obtained frequency distributions were compared with other previously reported population data.     

Genetic polymorphisms of 20 STR loci in Chinese Han population in Tianjin,North China

The PowerPlex^® 21 System PCR Amplification Kit was a new PCR Amplification Kit developed for forensic laboratories, but there was a lack of data about this kit in Chinese people in Tianjin, North China. This kit contained 20 STR loci, D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433 and FGA. In order to evaluate this kit and to get basic population data for its use in forensic practice in Chinese Han population, 360 unrelated Chinese Han individuals from Tianjin were typed using the Kit. Allele frequencies of the 20 STR loci and further population forensic genetic parameters were obtained. The observed genotype frequencies and expected genotype frequencies were evaluated by χ² test. No significant deviation from the Hardy–Weinberg equilibrium was observed in the population sample for the 20 STR loci. The population data in the present study can be used for routine forensic practice in Tianjin, North China.     

A search for obligatory paternal alleles in a DNA database to find an alleged rapist in a fatherless paternity case

Abstract: A sexual assault case resulted in a pregnancy, which was subsequently aborted. The alleged father of the fetus was unknown. Maternal and fetal types were obtained using the 11‐locus AmpF?STR^® SGM Plus^® kit. The national DNA database was searched for the paternal obligatory alleles and detected two suspects who could not be excluded as father of the male fetus. Additional typing using the AmpF?STR^® Minifiler^? kit, containing three additional autosomal loci, was not sufficient to exclude either suspect. Subsequent typing using the PowerPlex^® 16, containing four additional loci, and Y‐Filer^? kits resulted in excluding one suspect. Searching a database for paternal obligatory alleles can be fruitful, but is fraught with possible false positive results so that finding a match must be taken as only preliminary evidence.     

Texas Population Substructure and Its Impact on Estimating the Rarity of Y STR Haplotypes from DNA Evidence*

Abstract: Three sampled populations of unrelated males—African American, Caucasian, and Hispanic, all from Texas—were typed for 16 Y short tandem repeat (STR) markers using the AmpFlSTR^® Yfiler^TM kit. These samples also were typed previously for the 13 core CODIS autosomal STR loci. Most of the 16 marker haplotypes (2478 out of 2551 distinct haplotypes) were observed only once in the data sets. Haplotype diversities were 99.88%, 99.89%, and 99.87% for the African American, Caucasian, and Hispanic sample populations, respectively. F_ST values were very small when a haplotype comprised 10–16 markers. This suggests that inclusion of substructure correction is not required. However, haplotypes consisting of fewer loci may require the inclusion of F_ST corrections. The testing of independence of autosomal and Y STRs supports the proposition that the frequencies of autosomal and Y STR profiles can be combined using the product rule.     

Developmental validation of the PowerPlex ESX 16 and PowerPlex ESX 17 Systems

We describe the developmental validation study performed on the PowerPlex^® ESX 16 (European Standard Extended 16) and the PowerPlex^® ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. The PowerPlex^® ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpFlSTR^® SGM Plus^® kit as standard size. The PowerPlex^® ESX 17 System amplifies the same loci as the PowerPlex^® ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52-95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25 pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

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 AmpF?STR^® 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.     

DNA Identification of Commingled Human Remains from the Cemetery Relocated by Flooding in Central Bosnia and Herzegovina

The floods in Bosnia and Herzegovina in May 2014 caused landslides all over the country. In the small village of ?eri?i, near the town of Zenica, a landslide destroyed the local cemetery, relocated graves, and commingled skeletal remains. As the use of other physical methods of identification (facial recognition, fingerprint analysis, dental analysis, etc.) was not possible, DNA analysis was applied. DNA was isolated from 20 skeletal remains (bone and tooth samples) and six reference samples (blood from living relatives) and amplified using PowerPlex^® Fusion and PowerPlex^®Y23 kits. DNA profiles were generated for all reference samples and 17 skeletal remains. A statistical analysis (calculation of paternity, maternity, and sibling indexes and matching probabilities) resulted in 10 positive identifications. In this study, 5 individuals were identified based on one reference sample. This has once again demonstrated the significance of DNA analysis in resolving the most complicated cases, such as the identification of commingled human skeletal remains.     

Applicability of the ParaDNA® Screening System to Seminal Samples

Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA^® Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework.     

Genetic characterization of a population from Cauca Department with 15 STRs markers

This study established allele frequencies and some parameters of forensic interest with 15 autosomal STRs markers in a sample of 172 unrelated individuals from the Department of Cauca – Colombia using the PowerPlex^® 16 BIO System (Promega CO) and Qiagen Multiplex PCR (Qiagen) kits. All markers analyzed showed more than 61% of heterozygosity. Penta E and Penta D were the only systems that are not in Hardy Weinberg equilibrium (p < 0.0033) after Bonferroni correction. The probabilities of paternity (W), the power of exclusion (PE) and of discrimination (PD) accumulated for all loci analyzed were 0.9999, 0.9999 and >0.9999, respectively. The parameters of forensic interest have values suitable for routine use in forensic genetics.     

A comparative study of two extraction methods routinely used for DNA recovery from simulated post coital samples

In sexual assault cases DNA profiling of spermatozoa can be of critical importance. Most methods use differential extraction of the spermatozoa to separate it from the female component. Here we have compared two commercially available differential extraction methods, the QIAamp^® DNA mini kit (Qiagen) and Differex™ with the DNA IQ^® System (Promega). Simulated postcoital samples were prepared using buccal cells from a female donor and spermatozoa from three male donors. A dilution series ranging from neat semen to a 1:1500 dilution (semen:dH₂O) was prepared and mixed with an equal volume of saliva from a female donor. Extraction efficiency was assessed using DNA concentration measured with NanoDrop 2000 and Quantifiler^® Human DNA Quantification Kit and the profile count of full, partial and mixed DNA profiles generated using SGM Plus and PowerPlex^® ESI 17. Statistical analysis was carried out using Randomisation in R, which is a robust model making no assumption of the distribution of data. Based on the amount of DNA extracted and the types of profiles no significant difference in the performance of the two extraction kits was seen. However, the processing time taken with the Differex™ System was about half than that of the QIAamp^® DNA mini kit and involved fewer liquid transfers.     

Comparison of two methods for isolating DNA from human skeletal remains for STR analysis

Abstract: The quality and efficiency of a standard organic DNA isolation method and a silica‐based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real‐time PCR, and STR profiles were obtained using the AmpFlSTR^® Identifiler^® PCR amplification kit. Overall, the silica‐based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica‐based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica‐based method does not improve neither the quality nor the quantity of DNA for STR profiling.