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Identity by descent (IBD) segments, uninterrupted DNA segments derived from the same ancestral chromosomes, are widely used as indicators of relationships in genetics. A great deal of research focuses on IBD segments between related pairs, while the statistical analyses of segments in irrelevant individuals are rare. In this study, we investigated the basic informative features of IBD segments in unrelated pairs in Chinese populations from the 1000 Genome Project. A total of 5922 IBD segments in Chinese interpopulation unrelated individual pairs were detected via IBIS and the average length of IBD was 3.71 Mb in length. It was found that 17.86% of unrelated pairs shared at least one IBD segment in the Chinese cohort. Furthermore, a total of 49 chromosomal regions where IBD segments clustered in high abundance were identified, which might be sharing hotspots in the human genome. Such regions could also be observed in other ancestry populations, which implies that similar IBD backgrounds also exist. Altogether, these results demonstrated the distribution of common background IBD segments, which helps improve the accuracy in pedigree studies based on IBD analysis.
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Pueblo Asiatico , Genoma Humano , Humanos , Pueblo Asiatico/genética , Genoma Humano/genética , Linaje , Proyectos de Investigación , ChinaRESUMEN
Insertion/deletion polymorphisms (InDels) are a category of highly prevalent markers in the human genome, characterized by their distinctive attributes, including short amplicon sizes and low mutation rates, which have shown great potential in forensic applications. Multi-allelic InDel and multi-InDel markers, collectively abbreviated as MM-InDels, were developed to enhance polymorphism by the introduction of novel alleles. Nevertheless, the relatively low mutation rates of InDels, coupled with the founder effect, result in distinct allele frequency distributions among populations. The divergent characteristics of InDels in different populations also pose challenges to the establishment of universally efficient InDel multiplex assays. To enhance the system efficiency of the InDel assay and its applicability across diverse populations, 39 MM-InDels with high polymorphism in five different ancestry superpopulations were selected from the 1000 Genomes Project dataset and combined with an amelogenin gender marker to construct a multiplex assay (named MMIDplex). The combined power of discrimination and the cumulative probability of exclusion of 39 MM-InDels were 1 - 1.3 × 10-23 and 1 - 9.83 × 10-6 in the Chinese Han population, and larger than 1-10-19 and 1-10-4 in the reference populations, relatively. These results demonstrate that the MMIDplex assay has the potential to obtain sufficient power for individual identification and paternity test in global populations.
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Genética Forense , Polimorfismo Genético , Humanos , Genética Forense/métodos , Frecuencia de los Genes/genética , Pueblo Asiatico , Mutación INDEL , Genética de Población , ChinaRESUMEN
Insertion/Deletion (InDel) polymorphic genetic markers are abundant in human genomes. Diallelic InDel markers have been widely studied for forensic purposes, yet the low polymorphic information content limits their application and current InDel panels remain to be improved. In this study, multi-allelic InDels located out of low complexity sequence regions were selected in the datasets from East Asian populations, and a multiplex amplification system containing 31 multi-allelic InDel markers and the Amelogenin marker (FA-HID32plex) was constructed and optimized. The preliminary study on sensitivity, species specificity, inhibitor tolerance, mixture resolution, and the detection of degraded samples demonstrates that the FA-HID32plex is highly sensitive, specific, and robust for traces and degraded samples. The combined power of discrimination (CPD) of 31 multi-allelic InDel markers was 0.999 999 999 999 999 999 85, and the cumulative probability of exclusion (CPE) was 0.999 920 in a Chinese Han population, which indicates a high discrimination power. Altogether, the FA-HID32plex panel could provide reliable supplements or stand-alone information in individual identification and paternity testing, especially for challenging samples.
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Dermatoglifia del ADN , Genética Forense , Humanos , Pueblo Asiatico/genética , Paternidad , Mutación INDEL , Genética de Población , Frecuencia de los GenesRESUMEN
The discrimination of body fluid stains provides crucial evidence during the investigation of criminal cases. Previous studies have demonstrated the practical value of mRNA profiling in body fluid identification. Conventional strategy of mRNA profiling entails reverse transcription and PCR amplification in two separate procedures with different buffer systems. In this study, we subjected the one-step multiplex reverse transcription PCR strategy to mRNA profiling with the inclusion of the same 18 tissue-specific biomarkers in the F18plex system targeting peripheral blood, menstrual blood, vaginal secretion, saliva, semen, and urine. The Qiagen OneStep RT-PCR kit and Titanium One-Step RT-PCR kit were applied to multiplex construction, while reproducible profiling results were obtained with both kits. Compared to the F18plex system, similar expression profiles of biomarkers were obtained in targeted tissues, while expected cross-reaction was observed in non-targeted body fluids. However, CYP2B7P1 and SPINK5 were detected in menstrual blood samples, which was not observed using the F18plex system. Full-profiling results were obtained in all samples using 0.1 ng peripheral blood and semen RNA, and 1 ng menstrual blood, vaginal secretion, saliva, and urine RNA. In conclusion, the application of one-step mRNA profiling strategy could be a reliable and economical method for the simplified, specific, and simultaneous analysis of tissue-specific biomarkers for the discrimination of body fluid origin.
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Líquidos Corporales/química , Perfilación de la Expresión Génica , Reacción en Cadena de la Polimerasa Multiplex/métodos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Biomarcadores/química , Femenino , Humanos , MasculinoRESUMEN
OBJECTIVES: To identify whether the relationship between Zhang A, Zhang B, Zhang C and Zhang X is the half-sibling relationship whose mother is sister (hereinafter referred to as the special half-sibling relationship) or the common first cousin relationship and discuss the application of ITO method in discriminating the special kinship. METHODS: DNA was extracted from blood stain of four identified individuals, PowerPlex® 21 System and AGCU 21+1 STR kit were used to detect autosomal STR genetic markers. Investigator® Argus X-12 QS kit was used to detect the X chromosome STR genetic markers, the special half-sibling index (SHSI) and first cousin index (FCI) and their likelihood ratio (LR) were calculated by ITO method. RESULTS: The LR results of SHSI to FCI, which were calculated based on autosomal STR genotyping and the analysis of X-STR genotyping results suggested that the relationship between Zhang A, Zhang B, Zhang C and Zhang X was inclined to be a special half-sibling relationship. CONCLUSIONS: For the identification of special kinship, it is necessary to comprehensively apply various genetic markers according to the case. After the conclusion that shared alleles cannot be excluded from the analysis, ITO method can be further used to establish discriminant assumptions according to the specific case to obtain objective and reliable identification opinions.
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Familia , Hermanos , Alelos , Dermatoglifia del ADN , Marcadores Genéticos , Genotipo , Humanos , Repeticiones de MicrosatéliteRESUMEN
BACKGROUND: An STR locus with tri-allelic pattern is occasionally observed in routine forensic casework. The extra copy of TPOX locus with tri-allelic pattern in populations has been assumed to be inserted into an X chromosome, which took place forth before the Bantu expansion in Africa. Nonetheless, the exact location of the duplication and the form of rearrangement in the human genome has not been clarified yet. RESULTS: In this study, we investigated the extra copy of type 2 tri-allelic pattern at TPOX in various populations. While allele 10 is the major third allele in Africa, allele 11 appears more frequent in America and overwhelming in Chinese and Korean populations, which might attribute to the population substructures. Results from the investigation of family cases showed that the transmission of the extra allele had a similar genetic pattern of autosomal genes. Furthermore, a whole-genome sequencing followed by bioinformatics analysis revealed that the intact form of chromosomal duplication and rearrangement occurred ~ 407 kb away from the authentic TPOX locus on chromosome 2 in two cases. The breakpoints of the insertion were further validated in most other tri-allelic subjects, which can imply the identical origin from the ancestral extra copy. Nevertheless, de novo chromosomal duplication and rearrangement at thyroid peroxidase gene occur in populations. CONCLUSIONS: Instead of the extra allele 10 in African populations, the main third allele at TPOX with tri-allelic pattern is allele 11 in Chinese and Korean populations. The insertion of the extra copy into chromosome 2 occurs in most subjects with tri-allelic pattern at TPOX and demonstrates the transmission of the third allele from parents to offspring. The breakpoints of the ancestral extra copy are defined, which shows evidence of its inheritance from African populations. In addition, the simple validation method would help improve tri-allelic pattern calling, distinguish de novo chromosomal rearrangements, and also count the frequencies among different geographic regions. Therefore, the statistical interpretation of tri-allelic pattern at TPOX could be enhanced during forensic practice.
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Alelos , Dosificación de Gen , Sitios Genéticos/genética , Reordenamiento Génico , Técnicas de Genotipaje , HumanosRESUMEN
The combination of immune checkpoint blockade (ICB) and chemotherapy has shown significant potential in the clinical treatment of various cancers. However, circulating regeneration of PD-L1 within tumor cells greatly limits the efficiency of chemo-immunotherapy and consequent patient response rates. Herein, we report the synthesis of a nanoparticle-based PD-L1 inhibitor (FRS) with a rational design for effective endogenous PD-L1 suppression. The nanoinhibitor is achieved through self-assembly of fluoroalkylated competitive peptides that target PD-L1 palmitoylation. The FRS nanoparticles provide efficient protection and delivery of functional peptides to the cytoplasm of tumors, showing greater inhibition of PD-L1 than nonfluorinated peptidic inhibitors. Moreover, we demonstrate that FRS synergizes with chemotherapeutic doxorubicin (DOX) to boost the antitumor activities via simultaneous reduction of PD-L1 abundance and induction of immunogenic cell death in murine colon tumor models. The nano strategy of PD-L1 regulation present in this study is expected to advance the development of ICB inhibitors and overcome the limitations of conventional ICB-assisted chemo-immunotherapy.
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Antígeno B7-H1 , Inmunoterapia , Humanos , Animales , Ratones , Ligandos , Apoptosis , Péptidos/farmacología , Línea Celular TumoralRESUMEN
The Y-chromosomal haplogroup tree, which consists of a group of Y-chromosomal loci with phylogenetic information, has been widely applied in anthropology, archaeology and population genetics. With the continuous updating of the phylogenetic structure, Y-chromosomal haplogroup tree provides more information for recalling the biogeographical origin of Y chromosomes. Generally, Y-chromosomal insertion-deletion polymorphisms (Y-InDels) are genetically stable as Y-chromosomal single nucleotide polymorphisms (Y-SNPs), and therefore carry mutations that can accumulate over generations. In this study, potential phylogenetic informative Y-InDels were filtered out in haplogroup O-M175, which is dominant in East Asia, based on population data retrieved from the 1000 Genomes Project. A group of 22 phylogenetic informative Y-InDels were identified and then assigned to their corresponding subclades of haplogroup O-M175, which provided a supplement for the update and application of Y-chromosomal markers. Especially, four Y-InDels were introduced to define subclades determined using a single Y-SNP.
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Obtaining a full short tandem repeat (STR) profile from a low template DNA (LT-DNA) still presents a challenge for conventional methods due to significant stochastic effects and polymerase slippage. A novel amplification method with a lower cost and higher accuracy is required to improve the DNA amount. Previous studies suggested that DNA polymerases without bypass activity could not perform processive DNA synthesis beyond abasic sites in vitro and our results showed a lack of bypass activity for Phusion, Pfu and KAPA DNA polymerases in this study. Based on this feature, we developed a novel linear amplification method, termed Linear Aamplification for double-stranded DNA using primers with abasic sites near 3' end (abLAFD), to limit the replication error. The amplification efficiency was evaluated by qPCR analysis with a result of approximately a 130-fold increase in target DNA. In a LT-DNA analysis, the abLAFD method can be employed as a pre-PCR. Similar to nested PCRs, primer sets used for the abLAFD method were designed as external primers suitable for commercial multiplex STR amplification assays. The practical performance of the abLAFD method was evaluated by coupling it to a routine PP21 STR analysis using 50 pg and 25 pg DNA. Compared to reference profiles, all abLAFD profiles showed significantly recovered alleles, increased average peak height and heterozygote balance with a comparable stutter ratio. Altogether, our results support the theory that the abLAFD method is a promising strategy coupled to STR typing for forensic LT-DNA analysis.
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ADN , Alelos , ADN/análisis , ADN/genética , Heterocigoto , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Ancestry inference through population stratification plays an important role in forensic applications. Specifically, ancestry information inferred from forensic DNA evidence can provide vital clues for criminal investigations. Current advances in ancestry inference mostly focus on ancestry informative markers. Hereinto, multi-InDel was proposed as one of the compound markers performing well in complex ancestral classification in the subpopulation of Asia. However, research on analytical methods necessary to make reliable predictions is lacking. The newly proposed compound markers could be assessed with alternative methods. In this study, promising discriminant methods were explored using multi-InDel markers for forensic ancestry inference. As a prerequisite, the adopted multi-InDel markers were assessed by classical methods for population genetics, such as FST analysis, MDS and STRUCTURE. In addition, dimensionality reduction methods and serial reduction strategies were applied for data visualization. Subsequently, machine learning methods, including logistic regression (LR), support vector machine (SVM), k-nearest neighbors (KNN) and extreme gradient boosting (XGBoost), were evaluated by diverse approaches. As the result of multifarious analyses through comparisons and estimations, XGBoost with one-hot encoding was shown to be more effective in population stratification and ancestry inference for challenging cases with admixed populations.
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Genética de Población , Mutación INDEL , ADN/genética , Frecuencia de los Genes , Humanos , Aprendizaje Automático , Polimorfismo de Nucleótido SimpleRESUMEN
Incorporating quantum dots (QDs) into dendritic mesoporous silica nanoparticles (DMSNs) for signal amplification of label materials represents an efficient strategy to improve the performance of lateral flow immunoassays (LFIAs). In this work, it is found that the CdSe/ZnS QD's size matters for balancing their loading amount and quantum yields (QYs) in the DMSNs-QDs based label materials and ultimately determining the performance of LFIA. The impacts of three CdSe/ZnS QDs with diameters of 9.1, 10.5 and 11.7 nm on CdSe/ZnS QDs incorporation and LFIA applications are studied. The increase of CdSe/ZnS QDs size from 9.1 to 11.7 nm results in a decrease in CdSe/ZnS QDs loading amount and an increase in QYs of incorporated CdSe/ZnS QDs. This trade-off leads to an optimized CdSe/ZnS QDs size of 10.5 nm, which exhibits the best LFIA performance due to the balanced QDs loading (2.26 g g-1) and QY (57.1%). The 10.5 nm CdSe/ZnS QDs incorporated DMSNs-QDs for C-reactive protein (CRP) detection achieved a limit of detection of 5 pg mL-1 (equivalent to 4.2 × 10-14 M) with naked eye, which is lower than literature reports and commercial LFIA products. This study demonstrates that the CdSe/ZnS QD's size matters for improving the quality of DMSNs-QDs and their LFIA performance for CRP determination, providing new insights into the rational design of advanced label materials for improving LFIA performance.
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Técnicas Biosensibles , Compuestos de Cadmio , Puntos Cuánticos , Compuestos de Selenio , Proteína C-Reactiva , Inmunoensayo , Sulfuros , Compuestos de ZincRESUMEN
Y chromosome has an important role in the forensic practice due to its unique paternal inheritance pattern. Y-chromosomal single nucleotide polymorphisms (Y-SNPs) could provide supplementary information while the application of Y-chromosomal STR (Y-STR) haplotypes encounter their limitations. Y-SNPs with recurrent mutation can be seen in different Y-chromosomal haplogroups, which might help discriminate different paternal pedigrees. In this study, a host of candidate Y-SNPs with recurrent mutation were obtained based on population data from 1000 Genome Project. Further, 8 Y-SNPs from a small part of candidates were confirmed to be polymorphic in 2 or more Y-chromosomal haplogroups (sub-haplogroups) in the Chinese Han population. With a haplotype diversity value of 0.9367, the investigated subset of Y-SNPs with recurrent mutation shows a high discrimination power. Therefore, Y-SNPs with recurrent mutation should function as useful markers to provide information in the forensic applications.
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Cromosomas Humanos Y , Polimorfismo de Nucleótido Simple , Genética de Población , Haplotipos , Humanos , Repeticiones de Microsatélite , MutaciónRESUMEN
Rationally designed carbon materials with superstructures are promising candidates in applications such as electrocatalysis. However, the synthesis of highly porous carbon superstructures with macropores and carbon defects from a simple crystalline solid remains challenging. In this work, superstructured macroporous carbon rods composed of defective graphitic nanosheets are synthesized by direct carbonization of crystalline poly tannic acid (PTA) rods as precursors. During carbonization, PTA rods with a highly ordered lamellar structure induce a spatially confined two-step localized contraction that takes place in different dimensions and directions in each step. The unexpected contraction behavior results in the sponge-like macroporous carbon superstructure with large surface area, high porosity, and abundant defects, thus showing a superior electrocatalytic performance with high activity and selectivity for oxygen reduction reaction. The study provides new understandings in the design of functional carbon materials with distinctive structures and applications.
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The calculation of the paternity index (PI) value of common bi-allelic genotypes at STR loci has been standardized in paternity cases. However, for tri-allelic patterns, a rare category of genotyping aberration in forensic practice, the statistical analysis in paternity testing remains disputed. The Type 1 tri-allelic pattern generally results from somatic mutation in the early stage of individual development. The Type 2 tri-allelic pattern is commonly generated by segmental duplication in the genome. In this study, practical and theoretical aspects of the evaluation of evidence concerning the Type 1 and Type 2 tri-allelic patterns in healthy individuals are discussed based on the likelihood ratio (LR) in different categories of kinship cases. The calculation of the PI value concerning tri-allelic genotypes is formulated according to the generation and genetic transmission of tri-allelic patterns. Meanwhile, a package tool named TriPI is developed to assist the calculation of the PI value in paternity testing concerning tri-allelic subjects, which could benefit the evaluation of the weight of evidence in the interpretation of tri-allelic pattern in forensic practice.
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Alelos , Repeticiones de Microsatélite , Modelos Estadísticos , Paternidad , Humanos , Funciones de Verosimilitud , Masculino , Duplicaciones Segmentarias en el GenomaRESUMEN
BACKGROUND: D5S818 discrepancies have been reported in forensic parental testing due to null alleles. However, more cases may be ignored since proportional null alleles were missed without detection of heredity discrepancy between parents and offspring. RESULTS: In this study, null allele 12 at D5S818 was detected by the PowerPlex® 21 System with a higher occurrence rate on the basis of review on 2824 samples from the 1282 routine cases in Chinese Han population. Sequencing results revealed novel variant of guanine (G) into adenine (A) in the 7th [AGAT] repeats in the core repeat region accompanied by rs1187948322 in the samples with null allele 12. CONCLUSIONS: Forensic STR typing may benefit from this discovery: (1) primer design of CE profiling system could be improved for sensitive population and (2) polymorphic information could be enriched for the accuracy and precision of NGS genotyping system. Peak area of D5S818 was also analyzed through different commercial STR kits. It is suggested that more attention should be paid on observed homozygosity with reduced peak area, especially for the samples from Chinese Han population.
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Genética Forense/métodos , Pruebas Genéticas/métodos , Polimorfismo Genético , Análisis de Secuencia de ADN/métodos , Adulto , Niño , Femenino , Genética Forense/normas , Pruebas Genéticas/normas , Humanos , Masculino , Repeticiones de Microsatélite , Linaje , Juego de Reactivos para Diagnóstico/normas , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/normasRESUMEN
Multiple mutational events of insertion/deletion occurring at or around InDel sites could form multi-allelic InDels and multi-InDels (abbreviated as MM-InDels), while InDels with random DNA sequences could imply a unique mutation event at these loci. In this study, preliminary investigation of MM-InDels with random sequences was conducted using high-throughput phased data from the 1000 Genomes Project. A total of 3,599 multi-allelic InDels and 6,375 multi-InDels were filtered with multiple alleles. A vast majority of the obtained MM-InDels (85.59%) presented 3 alleles, which implies that only one secondary insertion or deletion mutation event occurred at these loci. The more frequent presence of two adjacent InDel loci was observed within 20 bp. MM-InDels with random sequences presented an uneven distribution across the genome and showed a correlation with InDels, SNPs, recombination rate, and GC content. The average allelic frequencies and prevalence of multi-allelic InDels and multi-InDels presented similar distribution patterns in different populations. Altogether, MM-InDels with random sequences can provide useful information for population resolution.
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The application of X-chromosomal short tandem repeats (X-STRs) has been recognized as a powerful tool in complex kinship testing. To support further development of X-STR analysis in forensic use, we identified nine novel X-STRs, which could be clustered into three linkage groups on Xp21.1, Xq21.31, and Xq23. A multiplex PCR system was built based on the electrophoresis. A total of 198 unrelated Shanghai Han samples along with 168 samples from 43 families was collected to investigate the genetic polymorphism and forensic parameters of the nine loci. Allele numbers ranged from 5 to 12, and amplicon sizes ranged from 146 to 477 bp. The multiplex showed high values for the combined power of discrimination (0.99997977 in males and 0.99999999 in females) and combined mean exclusion chances (0.99997918 and 0.99997821 in trios, 0.99984939 in duos, and 0.99984200 in deficiency cases). The linkage between all pairs of loci was estimated via Kosambi mapping function and linkage disequilibrium test, and further investigated through the family study. The data from 43 families strongly demonstrated an independent transmission between LGs and a tight linkage among loci within the same LG. All these results support that the newly described X-STRs and the multiplex system are highly promising for further forensic use.
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In this work, Cu-Co bimetallic nanoparticles embedded carbon nanocubes (CuxCo10-x/CNC) are synthesized by direct carbonization of Cu-Co bimetal ZIF. The ratio of Cu and Co nanoparticles in CuxCo10-x/CNC as well as morphology, pore structure and graphitization degree of carbon substrates can be tuned by adjusting the molar ratio of Cu/Co (0:10, 1:9, 2:8, 3:7, 4:6 and 5:5) in ZIF precursors. The Fenton catalytic performances of CuxCo10-x/CNC are further studied by degrading a typical azo dye, Acid Orange II (AOII). The results show the CuxCo10-x/CNC with a Cu/Co ratio of 4/6 display the highest catalytic activity with faster dye degradation rate than other catalysts, which may be ascribed to the synergetic effects of optimized ratio of Cu/Co bimetals, high surface area and graphitized carbon framework. The stability and reusability of the catalyst has been investigated, showing a good performance after five consecutive runs. The catalysts prepared in this study can be used as an attractive alternative in heterogeneous Fenton chemistry and wastewater treatment.
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Y-chromosomal short tandem repeats (Y-STRs) are widely used in human research for the evaluation of population substructure or population differentiation. Previous studies show that several haplotype sets can be used for the evaluation of population differentiation. However, little is known about whether each Y-STR in these sets performs well during this procedure. In this study, a total of 20,927 haplotypes of a Yfiler Plus set were collected from 41 global populations. Different configurations were observed in multidimensional scaling (MDS) plots based on pairwise genetic distances evaluated using a Yfiler set and a Yfiler Plus set, respectively. Subsequently, 23 single-copy Y-STRs were characterized in the evaluation of population differentiation using the mean of allele frequency difference (mAFD) between populations. Our results indicated that DYS392 had the largest mAFD value (0.3802) and YGATAH4 had the smallest value (0.1845). On the whole, larger pairwise genetic distances could be obtained using the set with the top fifteen markers from these 23 single-copy Y-STRs, and clear clustering or separation of populations could be observed in the MDS plot in comparison with those using the set with the minimum fifteen markers. In conclusion, the mAFD value is reliable to characterize Y-STRs for efficiency in the evaluation of population differentiation.
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Cromosomas Humanos Y/genética , Variación Genética/genética , Genética de Población , Repeticiones de Microsatélite/genética , Dermatoglifia del ADN/métodos , Etnicidad/genética , Genética Forense , Frecuencia de los Genes , Haplotipos/genética , Humanos , MasculinoRESUMEN
Currently, mRNA profiling is widely investigated for forensic body fluid identification, while it is still required to advance the approach for those casework samples of limited quantity or low quality. The inclusion of circular RNAs (circRNAs) can facilitate the detection of mRNA markers in forensic body fluid identification. In this study, a multiplex assay for forensic body fluid identification (F18plex assay) was developed by incorporating 14 tissue-specific mRNA markers with circRNAs expression, 2 mRNA markers with high abundance and 2 housekeeping markers for the discrimination of the most common forensic body fluids, including blood, menstrual blood, saliva, vaginal secretion, semen and urine. The markers employed in the F18plex assay show similar specificity to previous reports. Additionally, even if all linear transcripts were completely erased, the expected markers in target biofluids could still be identified, which should help the discrimination of those aged biological stains. Results from sensitivity testing and the detection of mixtures demonstrate good sensitivity of the multiplex assay. Generally, full biomarker profiles could be obtained with ≥1 µl of blood, saliva, or semen, and ≥1 ng of total RNAs from menstrual blood, vaginal secretion, or urine samples, respectively, using this multiplex assay under the established conditions. Collectively, the newly established multiplex assay can assist in determining the biological origin of forensic stains.