Developmental validation of an mRNA kit: A 5-dye multiplex assay designed for body-fluid identification.

Identifying the sources of biosamples found at crime scenes is crucial for forensic investigations. Among the markers used for body fluid identification (BFI), mRNA has emerged as a well-studied marker because of its high specificity and remarkable stability. Despite this potential, commercially available mRNA kits specifically designed for BFI are lacking. Therefore, we developed an mRNA kit that includes 21 specific mRNA markers of body fluids, along with three housekeeping genes for BFI, to identify four forensic-relevant fluids (blood, semen, saliva, and vaginal fluids). In this study, we tested 451 single-body-fluid samples, validated the universality of the mRNA kit, and obtained a gene expression profile. We performed the validation studies in triplicates and determined the sensitivity, specificity, stability, precision, and repeatability of the mRNA kit. The sensitivity of the kit was found to be 0.1 ng. Our validation process involved the examination of 59 RNA mixtures, 60 body fluids mixtures, and 20 casework samples, which further established the reliability of the kit. Furthermore, we constructed five classifiers that can handle single-body fluids and mixtures using this kit. The classifiers output possibility values and identify the specific body fluids of interest. Our results showed the reliability and suitability of the BFI kit, and the Random Forest classifier performed the best, with 94% precision. In conclusion, we developed an mRNA kit for BFI which can be a promising tool for forensic practice.

From degraded to deciphered: ATAC-seq's application potential in forensic diagnosis.

In recent years, molecular biology-based diagnostic techniques have made remarkable strides and are now extensively utilized in clinical practice, providing invaluable insights for disease diagnosis and treatment. However, forensic medicine, especially forensic pathology, has witnessed relatively limited progress in the application of molecular biology technologies. A significant challenge in employing molecular techniques for forensic diagnoses lies in the quantitative and qualitative changes observed in diagnostic markers due to sample degradation-a recognized and formidable obstacle. Inspired by the success of DNA sequencing in forensic practices, which enables accurate individual identification even in cases involving degraded and deteriorated tissues and organs, we propose the application of the assay for transposase-accessible chromatin with sequencing (ATAC-seq) to identify targets at the transcriptional onset, exploring chromatin and DNA-level alterations for injury and disease inference in forensic samples. This study employs ATAC-seq to explore alterations in chromatin accessibility post-injury and their subsequent changes over a 2-h degradation period, employing traumatic brain injury (TBI) as a representative model. Our findings reveal high sensitivity of chromatin accessibility sites to injury, evidenced by shifts in thousands of peak positions post-TBI. Remarkably, these alterations remain largely unaffected by early degradation. Our results robustly endorse the notion that integrating and incorporating these specific loci for injury and disease diagnosis in forensic samples holds tremendous promise for practical application. We further validated the above results using human cortical tissue, which supported that early degradation did not significantly affect chromatin accessibility. This pioneering advancement in molecular diagnostic techniques may revolutionize the field of forensic science, especially forensic pathology.

Optimizing Analytical Thresholds for Low-Template DNA Analysis: Insights from Multi-Laboratory Negative Controls.

When analyzing challenging samples, such as low-template DNA, analysts aim to maximize information while minimizing noise, often by adjusting the analytical threshold (AT) for optimal results. A potential approach involves calculating the AT based on the baseline signal distribution in electrophoresis results. This study investigates the impact of reagent kits, testing quarters, environmental conditions, and amplification cycles on baseline signals using historical records and experimental data on low-template DNA. Variations in these aspects contribute to differences in baseline signal patterns. Analysts should remain vigilant regarding routine instrument maintenance and reagent replacement, as these may affect baseline signals. Prompt analysis of baseline status and tailored adjustments to ATs under specific laboratory conditions are advised. A comparative analysis of published methods for calculating the optimal AT from a negative signal distribution highlighted the efficiency of utilizing baseline signals to enhance forensic genetic analysis, with the exception of extremely low-template samples and high-amplification cycles. Moreover, a user-friendly program for real-time analysis was developed, enabling prompt adjustments to ATs based on negative control profiles. In conclusion, this study provides insights into baseline signals, aiming to enhance genetic analysis accuracy across diverse laboratories. Practical recommendations are offered for optimizing ATs in forensic DNA analysis.

Improving DNA mixtures analysis using compound markers composed of InDels and SNPs screened from the whole genome with next-generation sequencing.

Next-generation sequencing (NGS) allows for better identification of insertion and deletion polymorphisms (InDels) and their combination with adjacent single nucleotide polymorphisms (SNPs) to form compound markers. These markers can improve the polymorphism of microhaplotypes (MHs) within the same length range, and thus, boost the efficiency of DNA mixture analysis. In this study, we screened InDels and SNPs across the whole genome and selected highly polymorphic markers composed of InDels and/or SNPs within 300 bp. Further, we successfully developed and evaluated an NGS-based panel comprising 55 loci, of which 24 were composed of both SNPs and InDels. Analysis of 124 unrelated Southern Han Chinese revealed an average effective number of alleles (Ae ) of 7.52 for this panel. The cumulative power of discrimination and cumulative probability of exclusion values of the 55 loci were 1-2.37 × 10-73 and 1-1.19 × 10-28 , respectively. Additionally, this panel exhibited high allele detection rates of over 97% in each of the 21 artificial mixtures involving from two to six contributors at different mixing ratios. We used EuroForMix to calculate the likelihood ratio (LR) and evaluate the evidence strength provided by this panel, and it could assess evidence strength with LR, distinguishing real and noncontributors. In conclusion, our panel holds great potential for detecting and analyzing DNA mixtures in forensic applications, with the capability to enhance routine mixture analysis.

Single-nucleus transcriptomic mapping uncovers targets for traumatic brain injury.

The subventricular zone (SVZ) is a neurogenic niche that contributes to homeostasis and repair after brain injury. However, the effects of mild traumatic brain injury (mTBI) on the divergence of the regulatory DNA landscape within the SVZ and its link to functional alterations remain unexplored. In this study, we mapped the transcriptome atlas of murine SVZ and its responses to mTBI at the single-cell level. We observed cell-specific gene expression changes following mTBI and unveiled diverse cell-to-cell interaction networks that influence a wide array of cellular processes. Moreover, we report novel neurogenesis lineage trajectories and related key transcription factors, which we validate through loss-of-function experiments. Specifically, we validate the role of Tcf7l1, a cell cycle gene regulator, in promoting neural stem cell differentiation toward the neuronal lineage after mTBI, providing a potential target for regenerative medicine. Overall, our study profiles an SVZ transcriptome reference map, which underlies the differential cellular behavior in response to mTBI. The identified key genes and pathways that may ameliorate brain damage or facilitate neural repair serve as a comprehensive resource for drug discovery in the context of mTBI.

Single-nucleus transcriptomic mapping of blast-induced traumatic brain injury in mice hippocampus.

As a significant type of traumatic brain injury (TBI), blast-induced traumatic brain injury (bTBI) frequently results in severe neurological and psychological impairments. Due to its unique mechanistic and clinical features, bTBI presents diagnostic and therapeutic challenges compared to other TBI forms. The hippocampus, an important site for secondary injury of bTBI, serves as a key niche for neural regeneration and repair post-injury, and is closely associated with the neurological outcomes of bTBI patients. Nonetheless, the pathophysiological alterations of hippocampus underpinning bTBI remain enigmatic, and a corresponding transcriptomic dataset for research reference is yet to be established. In this investigation, the single-nucleus RNA sequencing (snRNA-seq) technique was employed to sequence individual hippocampal nuclei of mice from bTBI and sham group. Upon stringent quality control, gene expression data from 17,278 nuclei were obtained, with the dataset's reliability substantiated through various analytical methods. This dataset holds considerable potential for exploring secondary hippocampal injury and neurogenesis mechanisms following bTBI, with important reference value for the identification of specific diagnostic and therapeutic targets for bTBI.

Assessment of ForenSeq mtDNA Whole Genome Kit for forensic application.

Mitochondrial DNA (mtDNA) is an indispensable genetic marker in forensic genetics. The emergence and development of massively parallel sequencing (MPS) makes it possible to obtain complete mitochondrial genome sequences more quickly and accurately. The study evaluated the advantages and limitations of the ForenSeq mtDNA Whole Genome Kit in the practical application of forensic genetics by detecting human genomic DNA standards and thirty-three case samples. We used control DNA with different amount to determine sensitivity of the assay. Even when the input DNA is as low as 2.5 pg, most of the mitochondrial genome sequences could still be covered. For the detection of buccal swabs and aged case samples (bloodstains, bones, teeth), most samples could achieve complete coverage of mitochondrial genome. However, when ancient samples and hair samples without hair follicles were sequenced by the kit, it failed to obtain sequence information. In general, the ForenSeq mtDNA Whole Genome Kit has certain applicability to forensic low template and degradation samples, and these results provide the data basis for subsequent forensic applications of the assay. The overall detection process and subsequent analysis are easy to standardize, and it has certain application potential in forensic cases.

Development of specific and rapid detection of human DNA by recombinase polymerase amplification assay for forensic analysis.

The determination of human-derived samples is very important in forensic investigations and case investigation in order to determine vital information on the suspect and the case. In this study, we established a recombinase polymerase amplification (RPA) assay for rapid identification of human-derived components. The sensitivity of the assay was 0.003125 ng, with excellent species specificity, and human-derived DNA could be detected in the presence of non-human-derived components at a ratio of 1:1000. Moreover, the RPA assay had a strong tolerance to inhibitors, in the presence of 800 ng/µL humic acid, 400 ng/µL tannic acid, and 8000 ng/µL collagen. In forensic investigation, common body fluids (blood, saliva, semen, vaginal secretions) are all applicable, and the presence of DNA can be detected from samples after simple alkaline lysis, which greatly shortens the detection time. Four simulation and case samples (aged bones, aged bloodstains, hair, touch DNA) were also successfully applied. The above research results show that the RPA assay constructed in this study can be fully applied to forensic medicine to provide high sensitivity and applicability detection methods.

An MPS-Based 50plex Microhaplotype Assay for Forensic DNA Analysis.

Microhaplotypes (MHs) are widely accepted as powerful markers in forensic studies. They have the advantage of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), with no stutter and amplification bias, short fragments and amplicons, low mutation and recombination rates, and high polymorphisms. In this study, we constructed a panel of 50 MHs that are distributed on 21 chromosomes and analyzed them using the Multiseq multiple polymerase chain reaction (multi-PCR) targeted capture sequencing protocol based on the massively parallel sequencing (MPS) platform. The sizes of markers and amplicons ranged between 11-81 bp and 123-198 bp, respectively. The sensitivity was 0.25 ng, and the calling results were consistent with Sanger sequencing and the Integrative Genomics Viewer (IGV). It showed measurable polymorphism among sequenced 137 Southwest Chinese Han individuals. No significant deviations in the Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were found at all MHs after Bonferroni correction. Furthermore, the specificity was 1:40 for simulated two-person mixtures, and the detection rates of highly degraded single samples and mixtures were 100% and 93-100%, respectively. Moreover, animal DNA testing was incomplete and low depth. Overall, our MPS-based 50-plex MH panel is a powerful forensic tool that provides a strong supplement and enhancement for some existing panels.

Intermittent fasting ameliorates neuronal ferroptosis and cognitive impairment in mice after traumatic brain injury.

Ferroptosis, a newly characterized form of programmed cell death that results from lipid peroxidation and mitochondrial dysfunction, has been demonstrated to be involved in the pathogenesis of traumatic brain injury (TBI). Scientific evidence has shown that intermittent fasting (IF) reduces both the lipid peroxidation and the mitochondrial dysfunction, raising the question of whether IF affects the ferroptosis induced by TBI. Here, based on an established TBI animal model, we examine the effects of IF on the activation of ferroptosis pathway as well as related outcomes. We uncovered that a 1-mo IF elevated the protective Gpx4 and Hspb1 expression, and partly abolished the increase of Nfe2l2, Slc7a11, Alox8, Steap3, and Nox2 in the cortex, which were induced by TBI. Furthermore, the characteristic cellular damage induced by ferroptosis was alleviated by IF, as revealed by Perls' Prussian blue staining, Nissl staining, and transmission electron microscope examination. Consistently, we examined the outcomes of mice subjected to TBI and found an improved cognitive function of the IF mice. In sum, our study demonstrated, to our knowledge for the first time, that a 1-mo IF regimen partly ameliorates ferroptosis in the cortex of mice subjected to TBI, which potentially contributes to a lessening of cognitive impairment.

Single-nucleus profiling of adult mice sub-ventricular zone after blast-related traumatic brain injury.

Explosive blast-related traumatic brain injuries (bTBI) are common in war zones and urban terrorist attacks. These bTBIs often result in complex neuropathologic damage and neurologic complications. However, there is still a lack of specific strategies for diagnosing and/or treating bTBIs. The sub-ventricular zone (SVZ), which undergoes adult neurogenesis, is critical for the neurological maintenance and repair after brain injury. However, the cellular responses and mechanisms that trigger and modulate these activities in the pathophysiological processes following bTBI remain poorly understood. Here we employ single-nucleus RNA-sequencing (snRNA-seq) of the SVZ from mice subjected to a bTBI. This data-set, including 15272 cells (7778 bTBI and 7494 control) representing all SVZ cell types and is ideally suited for exploring the mechanisms underlying the pathogenesis of bTBIs. Additionally, it can serve as a reference for future studies regarding the diagnosis and treatment of bTBIs.

Corrigendum: A novel SNP-STR system based on a capillary electrophoresis platform.

[This corrects the article DOI: 10.3389/fgene.2021.636821.].

An overview of SNP-SNP microhaplotypes in the 26 populations of the 1000 Genomes Project.

Microhaplotypes (MHs) are a promising new type of forensic markers that are defined by the combinations of two- or more single-nucleotide polymorphisms (SNPs) within 200 bp. Their advantages, such as low mutation rates, lack of stutter artifacts, and short amplicons, have improved human identification, kinship analysis, ancestry prediction, and mixture deconvolution capabilities. Information on published MHs, e.g., allele frequencies, is available in widely used public databases, ALlele FREquency Database, and MicroHapDB. However, there are abundant non-published MHs spread over the whole genome, and those databases do not incorporate other databases (e.g., the SNP Database) to provide users with more integrated information. Therefore, it is essential to establish a robust, responsive, and comprehensive MHs database. In this study, we thoroughly screened for SNP-SNP MHs among 26 populations from the 1000 Genomes Project (Phase 3). All genotype data of SNPs in each MH were converted to PHASE input files, and allele frequencies were estimated using PHASE. We compiled a detailed summary of SNP-SNPs at the global, continental, and population levels focused on haplotypes and the Ae value and supplemented our database using dbSNP data (last updated in 2015). We have successfully established a dual-SNP MH database (D-SNPsDB) of MHs within 50 bp for 26 populations in the integration of basic data such as physical positions in the human genome, mapping of variant identifiers (rsIDs), allele frequencies, and basic variant information. For public database queries, the D-SNPsDB web app was developed with the R Shiny package to get integrated information.

Feasibility of using probabilistic methods to analyse microRNA quantitative data in forensically relevant body fluids: a proof-of-principle study.

Several studies have confirmed that microRNAs (miRNAs) are promising markers for body fluid identification since they were introduced to this field. However, there is no consensus on the choice of reference genes and identification strategies. In this study, 13 potential candidate miRNAs were screened from three forensically relevant body fluid datasets, and the expression of 12 markers in five body fluids was determined using a real-time quantitative method. Two probabilistic approaches, Naive Bayes (NB) and partial least squares discriminant analysis (PLS-DA), were then applied to predict the origin of the samples to determine whether probabilistic methods are helpful in body fluid identification using miRNA quantitative data. Furthermore, 14 reference combinations were used to validate the influence of different reference choices on the predicted results simultaneously. Our results showed that in the NB model, leave-one-out cross-validation (LOOCV) achieved 100% accuracy and the prediction accuracy of the test set was 100% in most reference combinations. In the PLS-DA model, the first two components could interpret about 80% expression variance and LOOCV achieved 100% accuracy when miR-92a-3p was used as the reference. This study preliminarily proved that probabilistic approaches hold huge potential in miRNA-based body fluid identification, and the choice of references influences the prediction results to a certain extent.

Role of p40phox in host defense against Citrobacter rodentium infection.

NADPH oxidase (NOX) is a membrane-bound enzyme complex that generates reactive oxygen species (ROS). Mutations in NOX subunit genes have been implicated in the pathogenesis of inflammatory bowel disease (IBD), indicating a crucial role for ROS in regulating host immune responses. In this study, we utilize genetically deficient mice to investigate whether defects in p40phox , one subunit of NOX, impair host immune response in the intestine and aggravate disease in an infection-based (Citrobacter rodentium) model of colitis. We show that p40phox deficiency does not increase susceptibility of mice to C. rodentium infection, as no differences in body weight loss, bacterial clearance, colonic pathology, cytokine production, or immune cell recruitment were observed between p40phox-/- and wild-type mice. Interestingly, higher IL-10 levels were observed in the supernatants of MLN cells and splenocytes isolated from infected p40phox -deficient mice. Further, a higher expression level of inducible nitric oxide synthase (iNOS) was also noted in mice lacking p40phox . In contrast to wild-type mice, p40phox-/- mice exhibited greater NO production after LPS or bacterial antigen re-stimulation. These results suggest that p40phox-/- mice do not develop worsened colitis. While the precise mechanisms are unclear, it may involve the observed alteration in cytokine responses and enhancement in levels of iNOS and NO.

A Novel SNP-STR System Based on a Capillary Electrophoresis Platform.

Various compound markers encompassing two or more variants within a small region can be regarded as generalized microhaplotypes. Many of these markers have been investigated for various forensic purposes, such as individual identification, deconvolution of DNA mixtures, or forensic ancestry inference. SNP-STR is a compound biomarker composed of a single nucleotide polymorphism (SNP) and a closely linked short tandem repeat polymorphism (STR), and possess the advantages of both SNPs and STRs. In addition, in conjunction with a polymerase chain reaction (PCR) technique based on the amplification refractory mutation system (ARMS), SNP-STRs can be used for forensic unbalanced DNA mixture analysis based on capillary electrophoresis (CE), which is the most commonly used platform in worldwide forensic laboratories. Our previous research reported 11 SNP-STRs, but few of them are derived from the commonly used STR loci, for which existing STR databases can be used as a reference. For maximum compatibility with existing DNA databases, in this study, we screened 18 SNP-STR loci, of which 14 were derived from the expanded CODIS core loci set. Stable and sensitive SNP-STR multiplex PCR panels based on the CE platform were established. Assays on simulated two-person DNA mixtures showed that all allele-specific primers could detect minor DNA components in 1:500 mixtures. Population data based on 113 unrelated Chengdu Han individuals were investigated. A Bayesian framework was developed for the likelihood ratio (LR) evaluation of SNP-STR profiling results obtained from two-person mixtures. Furthermore, we report on the first use of SNP-STRs in casework to show the advantages and limitations for use in practice. Compared to 2.86 × 103 for autosomal STR kits, the combined LR reached 7.14 × 107 using the SNP-STR method in this casework example.

Validation of the Microreader 40Y ID System: a Y-STR multiplex for casework and database samples.

Y-chromosome-specific short tandem repeat loci (Y-STRs) are commonly analysed in forensic science for paternity testing, familial searches, and, in sexual assault cases, to determine male DNA identity from mixed sources with high background female DNA content. The Microreader 40Y ID System is a six-dye multiplex amplification kit that contains 17 Y-STR loci from the Yfiler Plus PCR Amplification Kit and the powerplex Y23 system (DYS19, DYF385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS549, DYS635(Y GATA C4), DYS643, Y GATA H4, DYS460, DYS481, DYS533, DYF387S1, DYS449, DYS518, DYS570, DYS576, and DYS627), plus six high polymorphic loci (DYS444, DYS447, DYS557, DYS596, DYS527 a/b) as well as 4 additional candidate Y-STR loci (DYS593, DYF404S1, DYS645) and a Y-Indel loci (Rs2032678), thereby providing greater efficiency, compatibility, and accuracy. The Microreader 40Y ID System can directly amplify markers from blood or saliva on filter paper or FTA cards, without template extraction or purification, and can also be used for extracted DNA templates. To verify the efficiency and accuracy of the kit, the Microreader 40Y ID System was validated by investigating sensitivity, amplification conditions, male-male and male-female mixtures, PCR inhibition, species specificity, reproducibility, and efficacy with degraded samples. The Y-STR loci were also tested using 437 male samples from Tibet, Han, and Yi. The Microreader 40Y ID System was able to compensate for some of the shortcomings of Y-STR markers in practical applications, such as cost and profile interpretation, and fully meets the domestic Y chromosome database construction specifications and requirements.

Multi-Indel: A Microhaplotype Marker Can Be Typed Using Capillary Electrophoresis Platforms.

Since the concept of microhaplotypes was proposed by Kidd in 2013, various microhaplotype markers have been investigated for various forensic purposes, such as individual identification, deconvolution of DNA mixtures, or forensic ancestry inference. In our opinion, various compound markers are also regarded as generalized microhaplotypes, encompassing two or more variants in a short segment of DNA (e.g., 200 bp). That is, a set of variants (referred to herein as multi-variants) within a certain length includes single nucleotide polymorphisms (SNP), insertion/deletion polymorphisms (Indels), or short tandem repeat polymorphisms (STRs). At present, multi-variant is mainly aimed at multi-SNPs. However, the haplotype genotyping of multi-variants relies on single-strand analysis, mainly using massively parallel sequencing (MPS). Here, we describe a method based on a capillary electrophoresis (CE) platform that can directly obtain haplotypes of individuals. Several microhaplotypes consisting of three or more Indels with different insertion or deletion lengths in the range of less than 200 bp were screened out, each of which had at least three haplotypes. As a result, the haplotype of an individual was reflected by the length of its polymorphism. Finally, we established a multiplex amplification system containing 18 multi-Indel markers that could identify haplotypes on each chromosome of an individual. The combined power of discrimination (CPD) and the cumulative probability of exclusion (CPE) were 0.999999999997234 and 0.9984, respectively.

A new approach to detect a set of SNP-SNP markers: Combining ARMS-PCR with SNaPshot technology.

Microhaplotypes are a new promising type of forensic genetic marker. Without the interference of stutter and high mutation rates as for STRs, and with short amplification lengths and a higher degree of polymorphism than single SNP, microhaplotypes composed of two SNPs, SNP-SNP, have a strong application potential. Currently, the most common method to detect microhaplotypes is massive parallel sequencing. However, the cost and extensive use of instruments limit its wide application in forensic laboratories. In this study, we screened 23 new SNP-SNP loci and established a new detection method by combining a multiplex amplification refractory mutation system-based PCR (ARMS-PCR) and SNaPshot technology based on CE. First, we introduced an additional deliberate mismatch at the antepenultimate base from the 3' end of primers when designing ARMS-PCR for SNP 1 (the first SNP of the SNP-SNP). Then, single base extension primers for SNaPshot assay were designed next to the position of SNP 2 (the second SNP). Finally, 15 loci were successfully built into four panels and these loci showed a relatively high level of polymorphism in the Southwest Chinese Han population. All the loci had an average probability of informative genotypes (I value) of 0.319 and a combined discrimination power of 0.999999999. Therefore, this new detection system will provide a valuable supplement to current methods.