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.

Removal performance and mechanism for a three-dimensional electrode system treating biochemical effluent of a wastewater treatment plant.

With the increasingly strict discharge requirements, it is urgent for wastewater treatment plants (WWTPs) to find an efficient and feasible technology for advanced treatment. A three-dimensional (3D) electrode system was used to treat the real biochemical effluent of a WWTP collecting industrial and domestic wastewater in the present study. The 3D electrode system had the best performance at a current density of 2 mA/cm2 and an electrode distance of 3 cm. The kinetic analysis showed that the organic pollutant degradation conformed to pseudo-first-order kinetics. The COD removal of the 3D electrode system was more than twice that of the two-dimensional (2D) electrode system, and the energy consumption was 46.56% less than that of the 2D electrode system. By measuring the adsorption capacity and the electrocatalytic ability of the system to produce strong oxidizing species, it was demonstrated that granular activated carbon (GAC) had the synergy of adsorption and electrochemical oxidation, and ·OH playing the dominant role in oxidizing pollutants. At the same time, the organic contaminants adsorbed on GAC could be degraded. Finally, the adsorption-electrochemical oxidation mechanism was proposed. The above results highlighted that the 3D electrode system was a promising alternative method in the application of advanced treatment for WWTPs.

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.

Establishment of a co-analysis system for personal identification and body fluid identification: a preliminary report.

Analysis of genetic markers can provide clues for case investigation. Short tandem repeat (STR) detection and analysis are widely used for both personal identification and parentage testing. However, DNA analysis currently cannot provide sufficient information for body fluid identification. Tissue or cell sources of samples can be identified by detecting body fluid-specific mRNA markers, which have been studied thoroughly. Integrating STR profiling and mRNA expression patterns can provide more information than conventional methods for investigations and the reconstruction of crime scenes; this can be achieved by DNA/RNA co-extraction technology, which is economical, efficient, and suitable for low-template samples. Here, we propose a co-analysis system based on the PowerPlex 16 kit. This system can simultaneously amplify 25 markers, including 15 STRs, one non-STR amelogenin, and nine mRNA markers (three blood-specific, two saliva-specific, two semen-specific, and two housekeeping gene markers). The specificity and sensitivity of the co-analysis system were determined and aged and degraded samples were used to validate the stability of the co-analysis system. Finally, different DNA/RNA ratios and various carriers were evaluated. The results showed that the DNA/RNA co-analysis system correctly identified different types of body fluid stains. The STR profiles obtained using the co-analysis system were identical to those obtained using the PP16 kit, which demonstrates that the mRNA primers used did not affect STR profiling. Complete STR and mRNA profiles could be obtained from 1/8 portions of buccal swabs, 1/16 portions of swabs of blood and semen samples, 0.1 cm2 of blood samples, 0.25 cm2 of semen samples, and 1.0 cm2 saliva samples. Additionally, our findings indicate that complete STR and mRNA profiles can be obtained with this system from blood and semen samples when the DNA/RNA ratio is 1:1/32. This study suggests that the co-analysis system could be used for simultaneous personal identification and body fluid identification.

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.

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.

MHBase: A comprehensive database of short microhaplotypes for advancing forensic genetic analysis.

Microhaplotypes (MHs) were first recommended by Prof. Kidd for use in forensics because they can improve human identification, kinship analysis, mixture deconvolution, and ancestry prediction. Since their introduction, extensive research has demonstrated the advantages of MHs in forensic applications and provided useful data for different populations. Currently, two databases, ALFRED (ALlele FREquency Database) and MicroHapDB (MicroHaplotype DataBase), house the published MH information and population data. We previously constructed a single nucleotide polymorphism SNP-SNP MH database (D-SNPsDB) of MHs within 50 bp on the whole human genome for 26 populations integrating basic data such as physical genome positions, mapping of variant identifiers (rsIDs), allele frequencies, and basic variant information. Building upon the previous research, we further selected MHs containing at least two variants (SNPs and/or insertions/deletions [InDels]) within a short DNA fragment (≤ 50 bp) in 26 populations based on the 1000 Genomes Project dataset (Phase 3) to construct a more comprehensive database. Subsequently, we established a user-friendly website that allows users to search the MH database (MHBase) based on their research objectives and study population to find suitable loci and provides other functions such as querying reported loci, performing online calculations using the PHASE software, and calculating ancestral-related parameters. The loci in the database are classified as SNP-based MHs, which include only SNPs, and InDel-including MHs, which contain at least one InDel. Here, we provide a detailed overview of the MHBase and an analysis of shared loci at the global and continental levels, ancestral markers, the genetic distance within loci, and mapping with the genome annotation file. The website is an accessible and useful tool for researchers engaged in marker discovery, population studies, assay development, and panel design.

Base-metal oxide semiconductor electrodes for PPCP degradation: Ti-doped α-Fe2O3 for sulfosalicylic acid oxidation as an example.

Sulfosalicylic acid is a typical pharmaceutical and personal care product with high toxicity, environmental persistence, and low biodegradability. Electrochemical oxidation has been demonstrated to be a promising way for hazardous organics treatment, but it is severely limited by the high cost and resource shortage of electrode materials. Base-metal oxide semiconductor anodes have the merits of low cost, diversity, and tunable energy levels for charge transfer, and thus may be alternatives to the electrodes for wastewater treatment. Herein, we found that Ti-doped α-Fe2O3, as an example, could be efficient for sulfosalicylic acid oxidation, reaching comparable faraday efficiency of sulfosalicylic acid to that of the boron-doped diamond electrode. Ti-doped electrodes exhibited both higher removal rates and current efficiency compared to the undoped. This could be mainly ascribed to the enhanced charge transfer rate constant. Kinetic analysis shows that the apparent reaction order, in terms of sulfosalicylic acid in bulk solution, depended on applied potential and pollutant concentration. Mechanism study shows that the oxidation of sulfosalicylic acid was mainly through indirect pathway. Moreover, the oxidation products were determined and the oxidation mechanism was proposed. This study may open a door to employ base-metal oxide semiconductor anodes for the efficient treatment of organic wastewater.

Performance of alkali and Cu-modified ZSM-5 during catalytic ozonation of polyvinyl alcohol in aqueous solution.

A novel hierarchical Cu/ZSM-5 was prepared over alkaline treatment and incipient wet impregnation method for the catalytic ozonation of polyvinyl alcohol (PVA). Under the optimum preparation conditions, hierarchical Cu/ZSM-5 exhibited an excellent mineralization performance during the PVA degradation process, and the removal rate of TOC after 60 min of reaction was 47.86%, much higher than that of ozonation alone (5.40%). Its high catalytic activity could attribute to the large pore volume (0.27 cm3/g) and pore size (6.51 nm) which are beneficial for the distribution of loaded copper and adsorption performance for PVA. Compared to ·OH, 1O2 (2.66 times in 10 min) contributed more to the removal of PVA. The degradation of PVA was a combined process of direct ozone oxidation, catalytic ozonation and adsorption. With its high catalytic performance and stability, hierarchical Cu/ZSM-5 has a very broad application prospect in the process of catalytic ozonation of refractory pollutants.

Occurrence, removal, and risk assessment of emerging contaminants in aquatic products processing sewage treatment plants.

Emerging contaminants (ECs) in aquatic environments have attracted attention due to their wide distribution and potential ecotoxicities. Sewage treatment plants (STPs) are proven to be the major source of ECs in the aquatic environment, while there remains insufficient understanding of the removal and risk assessment of ECs in STPs. Here, we clarified the degradation and risk impact of 13 ECs in two aquatic product processing sewage treatment plants (APPSTPs) along the southeast coast of China. The concentrations of ECs followed the order: endocrine-disrupting chemicals (1877.85-15,398.02 ng/L in influent, 3.37-44.47 ng/L in effluent) > > sulfonamide antibiotics (SAs, 75.14-906.19 ng/L in influent, 1.14-15.33 ng/L in effluent) > pharmaceutical and personal care products (PPCPs, 44.47-589.93 ng/L in influent, 2.54-34.16 ng/L in effluent) ≈ fluoroquinolone antibiotic (54.76-434.83 ng/L in influent, 10.75-32.82 ng/L in effluent) > other antibiotics (16.21-51.96 ng/L in influent, 0.68-6.17 ng/L in effluent). Moreover, the concentrations of PPCPs (decreased by 55.33-87.65% in peak fishing season) and antibiotics (increased by 44.99% in peak fishing season) were affected by fishing activities. In particular, the sequencing batch reactor (SBR) process had a better removal effect than the anaerobic-anoxic-oxic (A2/O) process on the treatment of some contaminants (e.g., norfloxacin and nonylphenol). Risk evaluations of ECs demonstrated that nonylphenol and SAs were at high- and low-risk states, respectively. Overall, our results provide important information for the degradation treatment of ECs, which is essential for pollutant management policy formulation.

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.

Evaluation of microhaplotype panels for complex kinship analysis using massively parallel sequencing.

In recent years, microhaplotypes (MHs) have become a research hotspot within the field of forensic genetics. Traditional MHs contain only SNPs that are closely linked within short fragments. Herein, we broaden the concept of general MHs to include short InDels. Complex kinship identification plays an important role in disaster victim identification and criminal investigations. For distant relatives (e.g., 3rd-degree), many genetic markers are required to enhance power of kinship testing. We performed genome-wide screening for new MH markers composed of two or more variants (InDel or SNP) within 220 bp based on the Chinese Southern Han from the 1000 Genomes Project. An NGS-based 67plex MH panel (Panel B) was successfully developed, and 124 unrelated individual samples were sequenced to obtain population genetic data, including alleles and allele frequencies. Of the 67 genetic markers, 65 MHs were, as far as we know, newly discovered, and 32 MHs had effective number of allele (Ae) values greater than 5.0. The average Ae and heterozygosity of the panel were 5.34 and 0.7352, respectively. Next, 53 MHs from a previous study were collected as Panel A (average Ae of 7.43), and Panel C with 87 MHs (average Ae of 7.02) was formed by combining Panels A and B. We investigated the utility of these three panels in kinship analysis (parent-child, full siblings, 2nd-degree, 3rd-degree, 4th-degree, and 5th-degree relatives), with Panel C exhibiting better performance than the two other panels. Panel C was able to separate parent-child, full-sibling, and 2nd-degree relative duos from unrelated controls in real pedigree data, with a small false testing level (FTL) of 0.11% in simulated 2nd-degree duos. For more distant relationships, the FTL was much higher: 8.99% for 3rd-degree, 35.46% for 4th-degree, and 61.55% for 5th-degree. When a carefully chosen extra relative was known, this may enhance the testing power for distant kinship analysis. Two twins from the Q family (2-5 and 2-7) and W family (3-18 and 3-19) shared the same genotypes in all tested MHs, which led to the incorrect conclusion that an uncle-nephew duo was classified as a parent-child duo. In addition, Panel C showed great capacity for excluding close relatives (2nd-degree and 3rd-degree relatives) during paternity tests. Among 18,246 real and 10,000 simulated unrelated pairs, none were misinterpreted as a relative within 2nd-degree at a log10(LR) cutoff of 4. The panels presented herein could provide supplementary power for the analysis of complex kinship.

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.

Molecular Dietary Analysis of Three Sympatric Mustelidae in Northeast China.

Diet analysis is essential to fully understand the biology of a species and its function within the ecosystem, as well as being key in identifying food web interactions and the population dynamics of predators and prey. The understanding of the diet of small to mid-sized carnivores remains generally lacking or uninformative due to the inability for taxonomic resolution based on morphology. The yellow-throated marten (Martes flavigula), Eurasian river otter (Lutra lutra), and Siberian weasel (Mustela sibirica) are three important Mustelidae species in ecosystems of northeast China. Based on fecal DNA and a next-generation sequencing (NGS) approach, we analyzed the vertebrate prey of these three sympatric Mustelidae. Prey included 7 mammalian taxa, 10 fishes, 2 birds, and 2 amphibians, with 85% of the taxa assigned to the species level. In total, twenty-one vertebrate prey taxa were identified from seven yellow-throated martens, eight Eurasian river otters, and two Siberian weasels. Concerning identified dietary species, 10 taxa were consumed by yellow-throated martens, 14 by Eurasian river otters, and 4 by Siberian weasels. Some prey species were identified in more than one species. Amphibians and fishes were the most dominant Eurasian river otter prey categories, whereas Eurasian badger (Meles leucurus), birds, and rodents were the main yellow-throated marten prey; amphibians and rodents were largely contained in Siberian weasel prey. Among prey items, Dybowski's frog (Rana dybowskii) and Korean field mouse (Apodemus peninsulae) were identified in all three Mustelidae species but our analyses suggest potential diet preferences among Mustelidae species. Future studies should focus on understanding the trophic relationships of these three Mustelidae species, providing valuable information for their conservation planning.

Distribution characteristics of microplastics in typical organic solid wastes and their biologically treated products.

Enormous production, use, and disposal of plastic goods present great challenges to environmental sustainability. Microplastics (MPs) are added into land which may serve as a larger MP repository than the ocean. Organic solid wastes and their biologically treated products can easily enter the soil and accumulate in soil systems. The current work deals with the extraction, identification, and distribution of MPs in typical organic solid wastes (food waste, livestock manure, and sludge) and their biologically treated products. The ecological risks of MPs were also preliminarily evaluated. The results showed that the abundance of MPs in organic solid wastes was in order of sludge > food waste > livestock manure. The main categories of MPs were fibers and films including PE, PP, and PET. The colors of MPs were mainly black, red, blue, and green. MPs generally exhibited bulges, depressions, cracks, or holes after biological treatment, and would be degraded into smaller fragments with potentially greater ecological risks. It was also found that the polymer risk index (H) of MPs in semi-dynamic composting products, compound fertilizer, and biogas residue of sedimentation tank sludge were higher than 10, reflecting their high ecological risk. Thus, it is recommended that the input of (micro)plastics to organic solid wastes streams should be minimized and related management should be established for the utilization of organic solid wastes and their biologically treated products.

Animal Detection and Classification from Camera Trap Images Using Different Mainstream Object Detection Architectures.

Camera traps are widely used in wildlife surveys and biodiversity monitoring. Depending on its triggering mechanism, a large number of images or videos are sometimes accumulated. Some literature has proposed the application of deep learning techniques to automatically identify wildlife in camera trap imagery, which can significantly reduce manual work and speed up analysis processes. However, there are few studies validating and comparing the applicability of different models for object detection in real field monitoring scenarios. In this study, we firstly constructed a wildlife image dataset of the Northeast Tiger and Leopard National Park (NTLNP dataset). Furthermore, we evaluated the recognition performance of three currently mainstream object detection architectures and compared the performance of training models on day and night data separately versus together. In this experiment, we selected YOLOv5 series models (anchor-based one-stage), Cascade R-CNN under feature extractor HRNet32 (anchor-based two-stage), and FCOS under feature extractors ResNet50 and ResNet101 (anchor-free one-stage). The experimental results showed that performance of the object detection models of the day-night joint training is satisfying. Specifically, the average result of our models was 0.98 mAP (mean average precision) in the animal image detection and 88% accuracy in the animal video classification. One-stage YOLOv5m achieved the best recognition accuracy. With the help of AI technology, ecologists can extract information from masses of imagery potentially quickly and efficiently, saving much time.

Prevalence of Varied Coat Coloration in a Yellow-Throated Marten (Martes flavigula) Population.

Mammalian coat color is determined by heritable variations such as disease, nutrition, and hormone levels. Variation in animal coat color is also considered an environmental indicator and provides clues for the study of population genetics and biogeography. Records of abnormal coloration in the wild are rare, not only because it is often selected against, but also because of the difficulties in detection of the phenomenon. We used long-term camera-trapping data to first report abnormal coat coloration in yellow-throated marten (Martes flavigula) in China. Six types of abnormal coloration were found only in the Northeast Tiger and Leopard National Park, Northeast China, which were not reported in other regions in China. A total of 268 videos of Martes flavigula contained normal coloration, 455 videos of individuals of the species contained abnormal coloration, 437 contained the 'gloving' type (martens with de-pigmented front toes, paws or lower forelimbs), while the remaining other 18 videos contained five types (different degrees of white-spotting and dilution). The higher relative abundance index (0.428, 'gloving' to 0.329, normal) and wide distribution area of the 'gloving' type indicated that this abnormal coat coloration type is usual in Northeast China, which may reflect genetic variability in the local population. These records will contribute to further research on animal coat color and its corresponding adaptive strategy.

mRNA and microRNA stability validation of blood samples under different environmental conditions.

RNA molecules, including mRNAs and microRNAs (miRNAs), have been used for forensic body fluid identification. Specific body fluids present unique mRNA expression patterns, while miRNAs identifying body fluids are mainly differentially expressed. miRNAs are thought to be more stable than mRNAs, although this lacks adequate supporting data. In this study, we addressed perceived concerns regarding the stability of miRNAs and mRNAs in blood samples. The samples used in this study involved three groups. First, environmentally-degraded blood stain samples were exposed to a range of environmental conditions over 1-360 days to degrade naturally. Second, simulated-degraded samples were prepared using RNase A or high temperature (80 °C). Furthermore, two authentic casework samples that were proven to be degraded from short tandem repeat (STR) profiles were analyzed. mRNAs and miRNAs present in the same blood samples were simultaneously detected through reverse transcriptase qPCR (RT-qPCR). Furthermore, mRNAs expression was determined by an mRNA multiplex PCR system. Our results showed that both mRNAs and miRNAs were stable in dry environments. The stability of miRNAs was relatively higher than that of mRNAs in humid environments or at high temperature. RNase A had the most serious impact on RNA stability, both mRNA profiles and miRNAs expression patterns were altered. The results of this study provide data and support to demonstrate that miRNAs represent more stable RNA molecules in body fluid identification compared to mRNAs.

Set of 15 SNP-SNP Markers for Detection of Unbalanced Degraded DNA Mixtures and Noninvasive Prenatal Paternity Testing.

Unbalanced and degraded mixtures (UDM) are very common in forensic DNA analysis. For example, DNA signals from criminal suspects are masked by a large amount of DNA from victims, or cell-free fetal DNA (cffDNA) in maternal plasma is masked by a high background of maternal DNA. Currently, detecting minor DNA in these mixtures is complex and challenging. We developed a new set of SNP-SNP microhaplotypes with short amplicons, and we successfully genotyped them using the new method of amplification-refractory mutation system PCR (ARMS-PCR) combined with SNaPshot technology based on a capillary electrophoresis (CE) platform. This panel reflects a high polymorphism in the Southwest Chinese Han population and thus has excellent potential for mixture studies. We evaluated the feasibility of this panel for UDM detection and noninvasive prenatal paternity testing (NIPPT). Fifteen SNP-SNPs detected minor DNA of homemade DNA mixtures, with a sensitivity of 0.025-0.05 ng and a specificity of 1:1,000. In addition, the panel successfully genotyped degraded DNA from single and mixed samples. Finally, 15 SNP-SNPs were applied to 26 trios. All samples displayed positive results with at least one marker to detect cffDNA. Besides, all fetal alleles in maternal plasma were confirmed by genotyping fetal genomic DNA from amniocentesis and paternal genomic DNA from peripheral blood. The results indicated that the SNP-SNP strategy based on the CE platform was useful for UDM detection and NIPPT.