Developmental Validation of AmpFℓSTR® Identifiler® Plus Kit for Forensic Applications

High-speed and high-accuracy DNA sequencing by capillary gel electrophoresis in a simple, low cost instrument Two-color peak-height encoded sequencing at 40°C

This paper reports a technique for detecting the presence of DNA in real-time PCR using label-free electrochemical impedance spectroscopy. The change in the real and imaginary parts of the impedance with the number of PCR cycles indicates the concentration of amplified DNA. In general, the real part of the impedance tends to increase with the number of PCR cycles over most of the measurement frequency range (100 Hz–1 MHz), whereas the imaginary part of the impedance increases in the negative direction. The optimal frequency for impedance analysis is determined by the performance index (PI), as one of the figures of merit, which quantitatively indicates how monotonously the impedance changes with the PCR cycle number. The detectability of DNA in the sample is scrutinized by analyzing the impedance at the optimal frequency (3.984 kHz and 20.02 kHz for the real and imaginary parts of the impedance, respectively) obtained from the performance index. The proposed technique can be used to quantitatively analyze PCR by applying simple electrochemical impedance spectroscopy (EIS) technology so that a label-free real-time monitoring system has high potential for a portable PCR system.

Forensic DNA analysis has revolutionized criminal investigations by providing valuable insights into identifying perpetrators, exonerating the innocent and unravelling mysteries of the past. Various molecular biology techniques, such as Short Tandem Repeat (STR) analysis, Single Nucleotide Polymorphism (SNP) profiling, mitochondrial DNA (mtDNA) sequencing, epigenetics and DNA methylation analysis, have played crucial roles in forensic investigations. While these techniques offer exceptional sensitivity and specificity, they also come with unique challenges. This review explores the current approaches of forensic DNA analysis, highlighting each technique. STR analysis, regarded as the gold standard in forensic DNA profiling, provides high discrimination power. However, it is limited in its ability to analyze degraded or mixed samples. On the other hand, SNP profiling offers advantages in analyzing highly degraded DNA samples but lacks the discriminatory power of STRs. Meanwhile, mtDNA analysis, particularly useful in cases involving compromised nuclear DNA, presents challenges due to its maternal inheritance pattern and lower discriminatory power. Furthermore, explore the exciting realm of epigenetics and the analysis of DNA methylation in forensic investigations. Epigenetic markers offer insights into gene expression patterns influenced by environmental factors, potentially aiding in the determination of tissue origin and chronological age estimation. DNA methylation analysis holds promise in forensic applications, providing additional layers of information for identity verification and tissue differentiation. Despite these advancements, several challenges persist in forensic DNA analysis, including the interpretation of complex DNA mixtures, standardization of methodologies, ethical considerations, and privacy concerns associated with the use of genetic information. Moreover, the integration of multi-omics data and machine learning approaches presents both opportunities and challenges in enhancing the accuracy and reliability of forensic DNA analysis. Looking ahead, future directions in molecular biology research for forensic DNA analysis involve the development of novel techniques with increased sensitivity, scalability, and robustness. By addressing these challenges and embracing emerging technologies, the field of forensic DNA analysis is poised for further advancements, offering enhanced capabilities in criminal justice and humanitarian efforts.

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit, developed and supplied by Applied Biosystems, complements the AmpFlSTR Identifiler polymerase chain reaction amplification kit (Applied Biosystems, Warrington, U.K.) by improving the success rate when profiling DNA that is degraded or contains inhibitors. Before applying the MiniFiler kit to casework, the profiles from 200 unrelated Kuwaitis were compared to Identifiler profiles. Concordance was observed for 99.875% (1598 of 1600) of the compared STR loci. The two discordant profiles displayed allelic dropout: one at the D13S317 locus due to nonamplification of allele 10 in the MiniFiler profile, and one at the D18S51 locus due to nonamplification of allele 18 in the Identifiler profile.

Biological evidence of sexual assault is one of the most difficult sample types to analyze in forensic laboratories. Y-STR markers are thus a valuable tool for analyzing these samples. The aim of this project was to compare three Y-STR commercial kits by analyzing their amplification performance on casework samples. Overall, 247 trace samples were analyzed with a Yfiler® Plus PCR Amplification Kit (Applied Biosystems), PowerPlex® Y23 (Promega® ) System and AmpFLSTR® Yfiler™ PCR Amplification Kit (Applied Biosystems). Comparing the amplification performance of the three kits, the first two were significantly more sensitive than the latter (p < 0.001). For samples, with a male DNA quantity less than 0.5 ng, the PowerPlex Y23® kit was the most sensitive and best performing kit, followed by the Yfiler® Plus kit (p = 0.009). In conclusion, the Yfiler® Plus and PowerPlex Y-23® kits are viable alternatives to older kits for samples with low amounts of male DNA.

Multiplex STR typing of aortic tissues from unidentified cadavers

A validation study was performed for the AmpFlSTR Yfiler PCR Amplification Kit (the Yfiler kit) to evaluate its potential applicability to Y-STR analysis in forensic casework. The optimal DNA input for PCR amplification was approximately 1 ng of male DNA. A marker-specific stutter ratio to filter stutter peaks higher than that defined by the kit manufacturer has been adopted for some markers to more readily and efficiently type Japanese male samples. No non-specific amplification was observed using 2000 ng female DNA as the PCR template. A full or partial DNA profile was detected from DNA mixture samples containing 1 ng of male DNA and 2000 ng of female DNA. From a mixture of two male DNA samples, all alleles possessed by the minor DNA contributor were detected when DNA derived from the minor contributor was present at 20% of the total DNA and above. Several loci were amplified when analyzing 23-year-old bloodstain samples derived from male individuals. Since they had not yet been defined by the kit manufacturer, we sequenced new alleles observed in Japanese males to assign accurate allele names. Analyses were also carried out of unusual alleles (an allele with poor amplification at DYS390 and apparent null alleles at DYS437 and DYS448) we observed in Japanese males. It is thought important that forensic biologists are aware of new and unusual alleles for DNA profile interpretation. Our validation study indicated the usefulness of the Yfiler kit for analyzing forensic biological samples containing male DNA.

Allele frequencies for 15 autosomal short tandem repeat (STR) loci included in the AmpFlSTR® Identifiler® PCR Amplification Kit from Applied Biosystems (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) and several statistical parameters were estimated from a sample of 224 unrelated individuals, mostly males, living in capital Baku (Azerbaijan) from central and southern governorates. The allele frequency spectrum detected in the Azerbaijani sample were compared to allele frequencies from 5 other data sets from published studies of individuals from Turkey, Iran, Russian, Caucasians and American populations. Significant global differences in allele frequencies were detected. Key words: Azerbaijan population, short tandem repeat DNA typing (STR DNA typing), population data, allele frequencies.

Austroasiatic (AA) speakers constitute around 4% of the population of Thailand, while the majority (89.4%) speak Kra-Dai (KD) languages. Previous forensic and population genetic studies in various Thai populations have employed a limited number of short tandem repeats (STRs). This study aims to expand the investigation of the genetic makeup of AA populations in Thailand and their relationship to KD populations using a larger number of autosomal STRs with the VeriFiler™ Plus PCR Amplification Kit. We generated 593 new genotypes from AA-speaking groups and combined them with previously reported data from AA and KD groups. A total of 1,129 genotypes across 23 STR loci were used to construct the largest allelic frequency profile for Thai and Lao populations. However, several loci deviated from Hardy-Weinberg equilibrium, likely due to the reduced genetic diversity in some highland populations, which should be considered in forensic investigations. Beyond forensic applications, our findings reveal genetic differences between AA-speaking groups in Northern and Northeastern Thailand. The AA groups from Northeastern Thailand exhibit greater genetic homogeneity and diversity, likely due to population interactions. In contrast, reduced diversity and increased heterogeneity in AA groups from Northern Thailand are possibly driven by genetic drift and cultural and geographic isolation. In conclusion, we emphasize the usefulness of increasing the number of autosomal STRs in forensic and anthropological genetic studies. Additional Y-STR and X-STR data from various AA-speaking groups in Thailand would further enhance and strengthen forensic STR databases in the region.

In spite of its shortcoming, analysis of PCR-derived rDNA libraries is being employed increasingly to investigate microbial diversity within many ecosystems. In the present investigation, the effects of the number of PCR cycles (10 vs 25 cycles) on the inferred structure of a 16S rDNA library have been examined. Seventy-five 25-cycle sequences were retrieved and analysed in comparison with 284 10-cycle sequences already described in a previous study. The 359 clones obtained were classified into 94 molecular species (at least 98% sequence similarity). At the level of large phylogenetic groups, the two cloned rDNA libraries were not different. A mathematical model was developed in order to estimate the number of molecular species expected if further sequencing was performed. Coverage-based computing, projections and statistical analysis demonstrated that the structures of the two PCR-derived rDNA libraries were different and that the 25-cycle rDNA library displayed reduced diversity. It is suggested that the number of PCR cycles used for amplification of 16S rDNA genes for phylogenetic diversity studies must therefore be kept as small as possible.

Evaluation of GeneMapper ®ID-X Mixture Analysis tool

Disaster victim identification (DVI) refers to the forensic identification of unknown individuals following a mass disaster event. Human dental structures can contain viable DNA sources when other soft tissues are compromised. However, labor-intensive sample preparation performed by extensively trained personnel is needed to expose the nuclear material for traditional forensic DNA workflows. With this in mind, we evaluated two simplified sample preparation protocols for processing tooth samples using either a conventional forensic DNA workflow or the Applied Biosystems® RapidHIT™ ID instrument. Briefly, sample sets for both protocols included 10 deciduous teeth that were cleaned prior to either fragmentation with a claw hammer (for RapidHIT™ ID processing) or fine-powder pulverization with a consumer-grade coffee grinder (for traditional workflows). The average percentage of expected STR alleles that were detected above analytical threshold for these tooth samples were comparable between methods: RapidHIT™ ID = 99.0% and GlobalFiler™ = 99.8%. Average intralocus heterozygote peak height ratios (PHRs) were comparable: RapidHIT™ ID = 0.80 and GlobalFiler™ = 0.86. Importantly, 9 of 10 samples analyzed via the RapidHIT™ ID required analyst review for flagged artifact peaks and quality issues. Across all profiles, 91% of alleles passed quality metrics for the RapidHIT™ workflow versus 100% for conventional GlobalFiler™ analysis. Collectively, these results suggest that quick, low-tech tooth sample fragmentation followed by analysis with the RapidHIT™ ID instrument can produce complete STR profiles from aged tooth samples. Future studies should include larger samples sets, more challenging tooth samples, and further simplification of sample preparation to enable field-forward, on-scene DVI.

The use of allelic imbalance to ascertain cis-regulation for human UGT2B7 in vivo

Droplet digital PCR improves urinary exosomal miRNA detection compared to real-time PCR

To the Editor: The European hedgehog, Erinaceus europaeus, is known to host a variety of tickborne pathogens, including the virus that causes tickborne encephalitis (1) and at least 3 species of the Borrelia burgdorferi sensu lato group: B. burgdorferi sensu stricto, B. afzelii, and B. garinii (2). Members of the B. burgdorferi s. l. group are the most common vectorborne pathogens of humans in central Europe (3). The role of hedgehogs as hosts for these pathogens is, therefore, of considerable epidemiologic interest. Hedgehogs are a common synanthropic species that live in urban, suburban, and rural environments (4) and are known to carry not only the hedgehog tick, Ixodes hexagonus, but also the most common European tick, I. ricinus (2,5). Both of these ticks are known vectors of B. burgdorferi s. l. and tickborne encephalitis virus; I. ricinus is the most important vector of both throughout Europe (1,5). To date, however, only limited information has been available on the role of the hedgehog as a host or reservoir for B. burgdorferi s. l. in Germany. We report the presence of 3 species of the B. burgdorferi s. l. group in European hedgehogs from Germany. To our knowledge, this is the first report of these species in hedgehogs in this country and the first report of B. spielmanii (A14S) (6) from this host. The investigated hedgehogs came from 2 sources: 9 from the ≈40 in an experimental plot in the city of Karlsruhe, state of Baden-Wuerttemberg, and the remainder from wild hedgehogs that had been brought to hedgehog care centers from various areas of Germany. All hedgehogs had died naturally, and tissue samples were taken from 43 animals (kidneys from 43, heart from 22, bladder from 33). The bodies had been frozen at –17°C before the samples were taken. DNA isolation was done by using the Maxwell 16 Instrument and System (Promega, Madison, WI, USA). Tissue samples were 3×3×3 mm. To detect B. burgdorferi s. l., we used 2 PCR protocols. The first was a nested PCR done according to the method of Rijpkema et al. (7). The target for the PCR was the spacer region between 5S and 23S rRNA genes of B. burgdorferi s. l. The nested primers generated a product of 226 bp. The amplified products were analyzed by agarose gel electrophoresis. The second protocol, a LightCycler-PCR hybridization assay (Roche Diagnostics, Mannheim, Germany) (8), simultaneously detects and genotypes the 3 genomic groups of B. burgdorferi s. l. This assay was specific for B. burgdorferi senso stricto, B. garinii, and B. afzelii (8) but also amplified B. spielmanii and B. valaisiana. The target for the PCR was the OspA gene. The PCR products of both systems were sequenced. For DNA sequencing reaction, the fluorescence-labeled didesoxynucleotide technology (Applied Biosystems, Darmstadt, Germany) was used. The sequenced fragments were separated, and the data were collected with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained sequences were then analyzed and compared by using BLAST (www.ncbi.nlm.nih.gov/BLAST). For 6 hedgehogs, Borrelia spp. could be clearly defined by using both gene sequences. Two additional animals had positive results, but sequencing was not possible because of either too little DNA or a mixed infection. B. spielmanii DNA was detected in the kidneys of 2 hedgehogs: 1 from Karlsruhe and 1 from 30 km west of this city in the German federal state of Rhineland-Palatinate. When sequences were compared by using BLAST, 4 BLAST sequences ({type:entrez-nucleotide,attrs:{text:AM055823,term_id:78482935,term_text:AM055823}}AM055823, {type:entrez-nucleotide,attrs:{text:AM055822,term_id:78482933,term_text:AM055822}}AM055822, {type:entrez-nucleotide,attrs:{text:DQ133518,term_id:74476812,term_text:DQ133518}}DQ133518, AY 995900) showed 100% similarity with B. spielmanii. B. garinii was detected in the heart of 2 animals (from Berlin and Karlsruhe); B. afzelii in 3 animals (in the kidney of 2 from Hamburg and Karlsruhe and in the bladder of 1 from Rhineland-Palatinate). A single animal (from Karlsruhe) had B. afzelii in the kidney and bladder and B. garinii in the heart. Preliminary results have also shown that ticks collected from hedgehogs from the Karlsruhe site were infected with B. afzelii (an I. hexagonus nymph and an I. ricinus female) and with B. spielmanii (an I. ricinus female, a nymph, and a larva) (Skuballa et al., unpub. data). These results show, that hedgehogs harbor at least 3 of the 5 recognized Borrelia genospecies found in Germany, all of which are known (B. afzelii, B. garinii) or are strongly suspected (B. spielmanii) of being pathogens for humans (9,10). To our knowledge, ours is the first report of B. spielmanii from hedgehogs, a Borrelia sp. that is usually associated with rodents, especially with garden and hazel dormice (10). That Borrelia spp. infections commonly occur in European hedgehogs is likely. However, questions remain about the role of these pathogens in regulating the populations of European hedgehogs and about the status of these common synanthropic mammals as a reservoir host of B. burgdorferi s. l. in periurban and rural environments.