Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

The Usefulness of qPCR Data for Sample Pre-Assessment and Interpretation of Genetic Typing Results.

DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction's nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant® (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex® ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs' peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile's characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders.

Looking into the Quantification of Forensic Samples with Real-Time PCR.

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/µL in the minimum extraction volume (40 µL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R2, and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event.

An Assessment of the Performance Limitations of the Integrated QuantifilerTM Trio-HRM Assay: A Forensic Tool Designed to Identify Mixtures at the Quantification Stage.

Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we integrated a high-resolution melt (HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated QuantifilerTM Trio-HRM assay. The assay, when coupled with a prediction tool, allowed for 75.0% accurate identification of the contributor status of a sample (single source vs. mixture). To elucidate the limitations of the developed qPCR-HRM assay, developmental validation studies were conducted assessing the reproducibility and samples with varying DNA ratios, contributors, and quality. From this work, it was determined that the integrated QuantifilerTM Trio-HRM assay is capable of accurately identifying mixtures with up to five contributors and mixtures at ratios up to 1:100. Further, the optimal performance concentration range was found to be between 0.025 and 0.5 ng/µL. With these results, evidentiary-like DNA samples were then analyzed, resulting in 100.0% of the mixture samples being accurately identified; furthermore, every time a sample was predicted as a single source, it was true, giving confidence to any single-source calls. Overall, the integrated QuantifilerTM Trio-HRM assay has exhibited an enhanced ability to discern mixture samples from single-source samples at the qPCR stage under commonly observed conditions regardless of the contributor's sex.

Detection of invisible biological traces in relation to the physicochemical properties of substrates surfaces in forensic casework.

Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person's skin's contact with an object or another person. Many factors influence touch DNA transfer, including the "destination" substrate's surface. The latter's physicochemical characteristics (wettability, roughness, surface energy, etc.) will impact touch DNA deposition and persistence on a substrate. We selected a representative panel of substrates from objects found at crime scenes (glass, polystyrene, tiles, raw wood, etc.) to investigate the impact of these characteristics on touch DNA deposition and detection. These were shown to impact cell deposition, morphology, retention, and subsequent touch DNA genetic analysis. Interestingly, cell-derived fragments found within keratinocyte cells and fingermarks using in vitro touch DNA models could be successfully detected whichever the substrates' physicochemistry by targeting cellular proteins and carbohydrates for two months, indoors and outdoors. However, swabbing and genetic analyses of such mock traces from different substrates produced informative profiles mainly for substrates with the highest surface free energy and therefore the most hydrophilic. The substrates' intrinsic characteristics need to be considered to better understand both the transfer and persistence of biological traces, as well as their detection and collection, which require an appropriate methodology and sampling device to get informative genetic profiles.

Dense single nucleotide polymorphism testing revolutionizes scope and degree of certainty for source attribution in forensic investigations.

The field of forensic DNA analysis has experienced significant advancements over the years, such as the advent of DNA fingerprinting, the introduction of the polymerase chain reaction for increased sensitivity, the shift to a primary genetic marker system based on short tandem repeats, and implementation of national DNA databases. Now, the forensics field is poised for another revolution with the advent of dense single nucleotide polymorphisms (SNPs) testing. SNP testing holds the potential to significantly enhance source attribution in forensic cases, particularly those involving low-quantity or low-quality samples. When coupled with genetic genealogy and kinship analysis, it can resolve countless active cases as well as cold cases and cases of unidentified human remains, which are hindered by the limitations of existing forensic capabilities that fail to generate viable investigative leads with DNA. The field of forensic genetic genealogy combined with genome-wide sequencing can associate relatives as distant as the seventh-degree and beyond. By leveraging volunteer-populated databases to locate near and distant relatives, genetic genealogy can effectively narrow the candidates linked to crime scene evidence or aid in determining the identity of human remains. With decreasing DNA sequencing costs and improving sensitivity of detection, forensic genetic genealogy is expanding its capabilities to generate investigative leads from a wide range of biological evidence.

Recovery of DNA from acetaminophen exploring physical state and sampling methods.

Research into the recovery of DNA from illicit drug samples has shown it is possible to get forensically useful profiles from such substrates. However, it is not yet known if the different physical states that drugs can be found in influences the quantity and quality of DNA that can be recovered or what is the best sampling method to adopt for powdered samples. This research used acetaminophen in four different states - large crystalline, powder, in solution, or residue - to determine the efficacy of current DNA technology in recovery and analysis of the resulting sample. Five replicates of each were prepared. Human blood was deposited on or mixed with the drug and left for 1 hour. The surface of the drug was sampled by wet/dry swabbing (where appropriate), or the entire sample was deposited in a tube, and the DNA then extracted using DNA-IQ™. The amount of DNA recovered (ng), degradation index, number of PCR cycles (Ct) required for the IPC to reach threshold, number of alleles in the DNA profile and average peak height (APH) were assessed. All samples, irrespective of the physical state they were collected from, returned full DNA profiles that corresponded to the DNA profile of the blood donor, with no degradation or inhibition detected. It was also found the wet/dry swabbing method returned higher levels of DNA than inclusion of the entire sample into the tube for powdered acetaminophen and the appropriate method to use will be dependent on casework circumstances. The findings of this research further develops our understanding of the recovery of DNA from drugs, and supports the need for further investigation to understand under what conditions DNA can be recovered from illicit substances.

Assessing DNA Degradation through Differential Amplification Efficiency of Total Human and Human Male DNA in a Forensic qPCR Assay.

The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of degradation. The possibility of deriving information on DNA degradation was evaluated in a forensic qPCR assay not specifically designed to detect DNA fragmentation, the Plexor HY (Promega), by calculating the ratio between the estimated concentrations of autosomal (99 bp) and Y-chromosomal (133 bp) targets ("[Auto]/[Y]"). The [Auto]/[Y] ratio measured in 57 formalin-fixed, paraffin-embedded samples was compared to a quality score (QS) calculated for corresponding STR profiles using quantitative data (allele peak height). A statistically significant inverse correlation was observed between [Auto]/[Y] and QS (R = -0.65, p < 0.001). The [Auto]/[Y] values were highly correlated (R = 0.75, p < 0.001) with the "[Auto]/[D]" values obtained using the PowerQuant (Promega) assay, expressly designed to detect DNA degradation through simultaneous quantification of a short (Auto) and a long (D) autosomal target. These results indicate that it is possible to estimate DNA degradation in male samples through Plexor HY data and suggest an alternative strategy for laboratories lacking the equipment required for the assessment of DNA integrity through dedicated qPCR assays.

Interpretation and reporting considerations of low-level DNA profiles following MinElute® purification of PowerPlex® 21 amplified products.

The generation of high-quality DNA profiles from trace amounts of DNA continues to be an issue in forensic casework. Several methods have been proposed over the years to increase recovery rates for low input DNA, including purification of PCR products, an increase in PCR cycle numbers and increasing injection time or voltage during electrophoresis. In this study, the characteristics of DNA profiles generated using QIAGEN MinElute® purification of Promega PowerPlex® 21 amplified products for low DNA input samples, ranging from 80 pg down to 4 pg, were evaluated. MinElute® purification was found to be a simple, effective and time efficient method, which can greatly improve the resolution of amplified PCR products, recovering 100% of donor concordant alleles from as little 16 pg of input template DNA and generating sufficient allelic information for interpretation from as low as 4 pg inputs. However, as is commonly observed with low template DNA samples, the results exhibited extensive disparity in the effects of stochastic variation in amplification, including increased heterozygote peak height imbalance, stutter ratios and instances of allelic drop-in and drop-out, both within and between replicates. As such, it is important that the extent and variability of these stochastic effects are appropriately incorporated in the development of robust profile interpretation guidelines for DNA profiles generated from purified PCR products.

The mysteries of DNA preservation in bone: A comparative study of petrous bones and metacarpal epiphyses using ATR-FTIR spectroscopy.

A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR typing. This unexpected parity in DNA preservation between the petrous bone, a renowned source of endogenous DNA in skeletal remains, and the epiphyses of metacarpals, which are porous and susceptible to taphonomic changes, is surprising. In this study, we introduced ATR-FTIR spectroscopy as an approach to unravel the correlation between bone molecular structure and DNA preservation. Metacarpals and petrous bones with same taphonomic history were sampled and prepared for DNA analyses. While one portion of the sample was used for DNA analysis, the other underwent ATR-FTIR spectroscopic examination. The normalized spectra and FTIR indices between the epiphyses of metacarpals and petrous bones were compared. Because the taphonomic history of the remains used is relatively short and stable, the ATR-FTIR spectroscopy unveiled subtle structural differences between the two bone types. Petrous bones exhibited higher mineralization, whereas epiphyses contained more organic matter. The unexpected preservation of DNA in the epiphyses of metacarpals can likely be attributed to the presence of soft tissue remnants within the trabeculae. Here observed differences in the molecular structure of bones indicate there are different mechanisms enabling DNA preservation in skeletal tissues.

Evaluation of post-PCR purification methods and subsequent optimisation of the MinElute® PCR Purification Kit for low input DNA samples amplified with PowerPlex® 21.

Weak and partial DNA profiles are commonly encountered within forensic casework due to amplification of low DNA input samples. One option for increasing allelic detection in such samples is the purification of amplified PCR product using commercially available column-based methods. In this study, four commercially available post-PCR purification methods, QIAGEN MinElute®, Independent Forensics Amplicon™ Rx, Millipore Microcon® and Thermo Fisher Scientific ExoSAP-IT™ were evaluated, comparing the quality of PowerPlex® 21 DNA profiles produced to the standard DNA profile generated prior to purification. An increased detection of alleles above the analytical threshold was observed following purification with the MinElute®, Amplicon™ Rx and Microcon® methods, allowing informative DNA profiles to be recovered using as little as 8 pg DNA. However, post-PCR purification using the ExoSAP-IT™ kit was unsuccessful, with no alleles detected above analytical threshold in samples with ≤16 pg DNA. The MinElute® kit was selected for optimisation on the basis of DNA profile quality, including increased detection of alleles and minimal artefacts. The MinElute® method was optimised by evaluating the number of washes and final elution buffer volume, resulting in a further increase in detection of alleles by reducing the elution buffer volume. Overall, this study showed that PowerPlex® 21 DNA profiles from low input DNA can be successfully enhanced by employing the MinElute® post-PCR purification method.

[Authenticity and adulteration identification of Bubali Cornu based on DNA fingerprints].

Whether adulteration exists is a difficult problem in the identification of traditional Chinese medicine(TCM). Bubali Cornu is mainly available in the medicinal material market in the form of buffalo horn silk or buffalo horn powder but lacks obvious identification characteristics, so there is a risk of adulteration. However, the method of identification of adulteration in Bubali Cornu is lacking at present. In order to ensure authenticity and identify adulteration of TCM Bubali Cornu, control the quality of TCM Bubali Cornu, and ensure the authenticity of clinical use, the DNA fingerprints of 43 batches of samples from pharmaceutical companies and medicinal material markets were identified, and the amplification primers of fluorescent DNA fingerprints of Bubali Cornu and Bovis Grunniens Cornu were screened. The DNA fingerprints of Bubali Cornu were obtained by fluorescent capillary typing. The identification effect of fluorescent capillary typing on different adulteration ratios was also tested. Two pairs of fluorescent STR typing primers, namely 16Sa and CRc, which can distinguish Bubali Cornu and Bovis Grunniens Cornu, were screened out, and a DNA fingerprint identification method was established. The 16Sa migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 223.4-223.9 bp and 225.5-226.1 bp. The CRc migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 518.8-524.8 bp and 535.9-542.5 bp. The peak height of the migration peak could be used for preliminary quantification of the adulterants with an adulteration ratio below 50%, and the quantitative results were similar to the adulteration ratio. In this study, a simple and quick universal DNA fingerprint method was established for the identification of Bubali Cornu and its adulterants, which could realize the identification of TCM Bubali Cornu and the semi-quantitative identification of the adulterants.

Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

PCR in Forensic Science: A Critical Review.

The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.

A DNA typing panel of 201 genetic markers for degraded samples: development and validation.

With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.

Biogeographical Ancestry Analyses Using the ForenSeqTM DNA Signature Prep Kit and Multiple Prediction Tools.

The inference of biogeographical ancestry (BGA) can assist in police investigations of serious crime cases and help to identify missing people and victims of mass disasters. In this study, we evaluated the typing performance of 56 ancestry-informative SNPs in 177 samples using the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx system. Furthermore, we compared the prediction accuracy of the tools Universal Analysis Software v1.2 (UAS), the FROG-kb, and GenoGeographer when inferring the ancestry of 503 Europeans, 22 non-Europeans, and 5 individuals with co-ancestry. The kit was highly sensitive with complete aiSNP profiles in samples with as low as 250pg input DNA. However, in line with others, we observed low read depth and occasional drop-out in some SNPs. Therefore, we suggest not using less than the recommended 1ng of input DNA. FROG-kb and GenoGeographer accurately predicted both Europeans (99.6% and 91.8% correct, respectively) and non-Europeans (95.4% and 90.9% correct, respectively). The UAS was highly accurate when predicting Europeans (96.0% correct) but performed poorer when predicting non-Europeans (40.9% correct). None of the tools were able to correctly predict individuals with co-ancestry. Our study demonstrates that the use of multiple prediction tools will increase the prediction accuracy of BGA inference in forensic casework.

Determining the effects of genetic linkage when using a combination of STR and SNP loci for kinship testing.

The pedigree likelihood ratio (LR) can be used for determining kinship in the forensic kinship testing. LR can be obtained by analyzing the DNA data of Short tandem repeat (STR) and single nucleotide polymorphism (SNP) loci. With the advancement of biotechnology, increasing number of genetic markers have been identified, thereby expanding the pedigree range of kinship testing. Moreover, some of the loci are physically closer to each other and genetic linkage between loci is inevitable. LRs can be calculated by accounting for linkage or ignoring linkage (LRlinkage and LRignore, respectively). GeneVisa is a software for kinship testing (www.genevisa.net) and adopts the Lander-Green algorithm to deal with genetic linkage. Herein, we used the simulation program of the software GeneVisa to investigate the effects of genetic linkage on 1st-degree, 2nd-degree, and 3rd-degree kinship testing. We used this software to simulate LRlinkage and LRignore values based on 43 STRs and 134 SNPs in commercial kits by using the allele frequency rate and genetic distance data of the European population. The effects of linkage on LR distribution and LRs of routine cases were investigated by comparing the LRlinkage values with the LRignore values. Our results revealed that the linkage effect on LR distributions is small, but the effect on LRs of routine cases may be large. Moreover, the results indicated that the discriminatory power of genetic markers for kinship testing can be improved by accounting for linkage.

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method.

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1-15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

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

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

Evaluation of metal ions and DNA recovery from the surface of fired and unfired brass ammunition to improve STR profiling.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence.