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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. •Short-term bloodstain age was estimated by the proportions of long DNA fragments.•Nanopore sequencing was applied to measure DNA fragment length.•The effect of water on DNA degradation was examined using bloodstains wetted once.

Disaster Victim Identification (DVI) following mass fatality events is critical in bringing closure to the victims’ family members and their loved ones. However, post-disaster environments are typically unfavourable and pose difficulties for the execution of DVI procedures. Delays in collecting, transporting, and processing the samples may cause DNA to degrade, adversely impacting the identification process. Therefore, this study aimed to demonstrate the co-utilisation of RapidHIT ID (RHID) and DJI Matrice 300 (M300) for onsite DNA analysis using buccal swab samples. 40 samples (two replicates) were collected from 20 “victims” and another 40 (two replicates) from 20 corresponding “relatives”. The first replicates were processed using RHID ( n  = 40) and the second replicates were analysed via conventional technologies ( n  = 40). This paper observed the genotyping success rate, kinship matching, concordance, comparing different sample collectors, storage interval, and time taken for both procedures. Results of this study showed that RHID could generate DNA profiles for all the samples ( n  = 40) with 90% of them showing full profiles, and managed to process samples that have been stored for up to six months. The drone-assisted procedure exhibits less time to obtain and analyse the samples but can still produce DNA profiles concordant with the conventional method ( p  > 0.05). In conclusion, RHID is sufficient to generate interpretable DNA profiles in harsh environments, and transporting samples by M300 drone can reduce the exposure time to process more quality DNA for DVI.

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.

The necessity for developing the AGCU EX-38 typing system arises from the ever-increasing demand for more accurate and comprehensive forensic tools. Traditional kits with fewer STRs often fall short in complex cases requiring higher resolution. The AGCU EX-38 typing system incorporates 35 autosomal STRs, including extended CODIS loci as well as additional non-CODIS loci (D6S1043, D19S3045, D3S3045, D7S3048, D11S2368, D4S2366, D8S1132, D15S659, Penta D, Penta E, D6S447, D3S1744, D14S608, D18S535). This combination of CODIS and non-CODIS markers provides a significant advantage, particularly in complex kinship analyses such as half-sibship cases. This six-dye kit encompasses 38 loci, with a maximum amplicon size of 550 base pairs (bp), and features nine STRs within 200 bp and 14 STRs within 300 bp, offering unparalleled coverage and sensitivity. The AGCU EX-38 typing system is the only available kit on the market containing 35 autosomal STRs with six-dye chemistry, making it a unique and invaluable resource for forensic laboratories. This configuration allows for higher resolution and superior performance in cases with degraded or mixed DNA samples. In this study, we report the results of the developmental validation study, which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines. The data includes PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design and demonstrate the kit’s robustness, reliability, and suitability for genetic identification and population studies.

Short tandem repeat (STR) kits, which enable the detection of genetic variations, similarities, and origins, are essential for forensic analysis. However, human-specific primers vary depending on the manufacturer, resulting in mismatched profiles, consequently limiting the kit efficiency. Efficient methods for acquiring primer information to elucidate the reasons behind the allelic discrepancies are yet to be developed. This study aimed to develop an analytical method for directly reading labeled PCR fragments using an Illumina next-generation sequencer. In this study, DNA libraries from seven commercial STR kits (GlobalFiler, GF; Yfiler Plus, YFP; AmpFLSTR Identifiler Plus, IDP; AmpFLSTR MiniFiler, MF; and AmpFLSTR Yfiler, YF; as well as the PowerPlex Fusion, PPF and PowerPlex 16 System, PP16) were sequenced into pair-end reads, and bioinformatic analysis was performed to predict the 5′ ends of the primer sequences in each kit. The 5′ ends of the primer sequences corresponding to peaks in the evaluated STR kits were further compared with published sequences to validate positional accuracy. The 5′ ends of primer pairs at all loci predicted in this study were perfectly matched to those of published primer pairs in the PP16 kits. Our method elucidated the reconstruction process from fluorescent peaks in the electropherogram into a sequence-based histogram for forensic massively parallel sequencing analysis. Moreover, the effect of single nucleotide polymorphism-specific primers in the GF was revealed. Our findings demonstrate that this method can be used to accurately identify the cause of mismatched profiles between kits or low amplification at a specific locus.

The ForenSeq™ DNA Signature Prep kit has not been thoroughly tested with crude buccal swab lysates in large-scale population studies using massively parallel sequencing (MPS). Commonly used lysis buffers for swabs intending to undergo direct polymerase chain reaction (PCR) are SwabSolution™ and STR GO! Lysis Buffers, and these have been successfully used to generate population data using capillary electrophoresis (CE) systems. In this study, we investigated the performance and optimisation of SwabSolution™ and STR GO! lysates with the ForenSeq™ DNA Signature Prep workflow and addressed the challenge of failed MPS profiles in initial trials. To mitigate PCR inhibition in SwabSolution™ lysates, three optimisation methods were evaluated: dilution of lysates, addition of 5X AmpSolution ® reagent, and purification with magnetic beads. For STR GO! lysates, we explored spin-column purification using the QIAamp ® DNA Investigator kit, magnetic bead purification, and a pH adjustment with 1 M hydrochloric acid. Our findings indicated that the addition of 5X AmpSolution ® was effective for overcoming PCR inhibition in SwabSolution™ lysates, thereby maintaining a direct PCR approach. Spin-column purification, however, is recommended for STR GO! lysates to minimise MPS profile failure rates. These improvements enhance first-time success rates of crude swab lysates, and reduce the need for repeat sampling and re-sequencing, making the workflow more suitable for large-scale population studies in forensic laboratories. Graphical Abstract Overview of the adaptations made to ensure a high-first time success rate with crude buccal swab lysates using the ForenSeq ™ DNA Signature Prep kit workflow

In recent years, DNA analysis techniques have drastically increased in sensitivity, allowing for low template DNA samples to be more easily detected and used for identification. Since the problems inherent in low template DNA are exacerbated in DNA mixture samples, it would be advantageous to incorporate an assay earlier in the DNA workflow that could detect a mixture and, potentially, determine the number of contributors. Some real-time PCR instruments have high-resolution melt curve analysis (HRM) capabilities, allowing for an opportunity to integrate an HRM screening assay into a commercial DNA quantification kit. This work describes the integration of a mixture screening HRM assay using STR loci D5S818 and D18S51 into Qiagen’s Investigator Quantiplex ® kit. The integrated Quantiplex ® -HRM assay was tested on two qPCR platforms: The Rotor-Gene ® Q and the QuantStudio™ 6 Flex. Data from this assay was analyzed using linear discriminant and support vector machine analyses for sample classification. When HRM curve data from the Rotor-Gene ® Q was used for classification, the integrated assay exhibited an overall accuracy of 89.39%, correctly classifying 87.5% of single source samples and 100% of mixtures. When HRM curve data from the QuantStudio™ 6 Flex was used for classification, the integrated assay exhibited an overall accuracy of 87.88%, correctly classifying 87.5% of single source samples and 90% of mixtures. The overall accuracy of the integrated Quantiplex ® -HRM assay on both instruments met our goal of ≥ 80% accuracy, demonstrating the viability of the assay to detect mixtures when integrated into a commercial quantification kit.

Massively parallel sequencing (MPS) overcomes many PCR-CE limitations to analyze STRs and allow simultaneous inclusion of SNPs in forensic cases. By MPS, the ForenSeq™ DNA Signature Prep kit analyzes 27 aSTRs, 7 X-STRs, 24Y-STRs, and 94 identity-informative SNPs (iiSNPs) with the DNA Primer Set-A (DPS-A). Optionally, the DNA Primer Set-B (DPS-B) adds to the analysis 56 ancestry-informative SNPs (aiSNPs) and 24 phenotype-informative SNPs (piSNPs), but diminishes from 96 to 32 the number of samples per sequencing run. We assessed the forensic informativity provided by the loci analyzed by these two DPS in admixed individuals from Mexico City (Center, Mexico). For STRs, we report length-based (LB) and sequence-based (SB) allele frequencies and forensic parameters of the 152 identity informative markers (DPS-A). For aSTRs, the combined PD of SB genotypes (PD ~ 100%) was ~ 2949 times larger than that from LB. Conversely, the observed phenotype distribution offered low PD levels (PD = 6.6% and 10.4%), whereas piSNPs predicted accurately only the modal brown eye and dark hair colors, respectively. Similarly, aiSNPs detected a large prevalence of admixed individuals (97.3%; PD = 5.4%). Although few individuals were inferred as Europeans and Native Americans (1.37% each), they were self-declared as admixed, which result confusing for HID purposes. In brief, SB genotypes increased significantly the informativity of STRs to solve complex cases (DPS-A), whereas aiSNPs and piSNPs added mostly irrelevant information (DPS-B). We provide useful cost-benefit criteria in one Latin American population to choose DPS-A (96 samples) instead of DPS-B (32 samples) of the Forenseq kit.

The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis.

The ForenSeq™ FGx System (Illumina, San Diego, CA) was initially evaluated in concordance with SWGDAM guidelines for internal validation to determine the quality of the system’s components: the ForenSeq™ DNA Signature Prep Kit reagents, the MiSeq FGx™ instrument, and the ForenSeq™ Universal Analysis Software, for the analysis of targeted, forensically informative single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). This multiplex consisted of STRs (autosomal, X, and Y) and SNPs (identity, ancestry, and phenotypic) that were run using one preparation process. Overall, the ForenSeq™ FGx System performed as well as the traditional capillary electrophoresis-based method in producing usable profile information, along with additional information that could aid in investigative leads. The MiSeq FGx™ System was validated using DNA samples in studies testing reproducibility, repeatability, concordance, sensitivity, and mock case single donor samples. Overall, genotyping results for STRs and SNPs were concordant with the profiles generated from conventional STR analysis using Identifiler and SNPs typed by 23andMe analysis. Genotypes of the ForenSeq™ aSNPs were used to evaluate biogeographical ancestry estimations using ForenSeq™ Universal Analysis Software, FROG-kb database (KIDD aiSNP 55 panel), and 23andMe. The system was shown to provide reproducible genotypes and reliable results were obtained at levels as low as 50 pg. All mock case samples were concordant with the donor profile. The results support consideration of the ForenSeq™ FGx System as an acceptable alternative to current STR and SNP analysis, pending formal developmental and internal validation studies.