Forensic Science International-Genetics最新文献

{"title":"An evaluation of molecular and statistical methods for the attribution of body fluid sources to donors","authors":"Anika Nina Correll Trnka ,&nbsp;Rebecca Richards ,&nbsp;Stephanie Opperman ,&nbsp;SallyAnn Harbison","doi":"10.1016/j.fsigen.2026.103455","DOIUrl":"10.1016/j.fsigen.2026.103455","url":null,"abstract":"<div><div>Attributing mixed biological materials in crime stains to individual donors remains a challenge in forensic science. Characterisation of the messenger RNA (mRNA) transcripts in a biological material can be used for body fluid identification (BFID) purposes. Comparing coding region single nucleotide polymorphisms (cSNPs) in mRNA transcripts obtained from crime stains to reference cSNP profiles from known individuals presents a way to assign biological materials to donors. In this study, saliva, blood, skin, and vaginal samples were collected from eleven participants. Based on previous studies, a sequencing assay was designed targeting 23 cSNPs in single source gDNA and RNA samples to determine the potential of using Illumina MiSeq sequencing technology for BFID and donor association with cSNPs. Promising results were obtained from this preliminary study. The RNA samples displayed expression patterns typical of the biological materials they were sourced from. The expression results were investigated with principal component analysis (PCA) and incorporated into a Bayesian Network (BN) for statistical interpretation, with encouraging results. Variant calling of cSNPs had modest success. While the results showed potential for donor association, the discriminatory power and sequencing quality of some RNA transcripts limit donor attribution prospects, especially for the saliva and skin samples.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"84 ","pages":"Article 103455"},"PeriodicalIF":3.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146260538","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HipSTR-UI: A cross-platform graphical interface for accessible str genotyping from next-generation sequencing data","authors":"Tamara Soledad Frontanilla ,&nbsp;Matheus de Sousa Ferrari ,&nbsp;Jesus Ayala ,&nbsp;Celso Teixeira Mendes-Junior","doi":"10.1016/j.fsigen.2026.103456","DOIUrl":"10.1016/j.fsigen.2026.103456","url":null,"abstract":"<div><div>Next-generation sequencing (NGS) has expanded the scope of forensic genetics by providing sequence-level resolution of Short Tandem Repeats (STRs). We developed HipSTR-UI, a cross-platform graphical interface that integrates precompiled HipSTR binaries for Windows, macOS, and Linux. The interface automates the genotyping workflow, from alignment files (BAM/CRAM) and target regions with the reference genome (FASTA) to the generation of genotypes in VCF format. HipSTR-UI includes a graphical parameter panel and exportable results. The validation was performed using Phase 3 of the 1000 Genomes Project, previously analyzed with the HipSTR command-line version. HipSTR-UI accurately reproduced the results of HipSTR, achieving 100 % concordance in genotype calls. As expected, no discrepancies were observed in concordance rates, discordant loci, or quality scores, since the interface executes the same commands as the original HipSTR tool. HipSTR-UI combines the robustness of HipSTR with a user-friendly and multilingual interface, bridging the gap between advanced sequencing technologies and routine forensic applications. Additionally, it facilitates adoption in routine forensic workflows, including human identification and kinship analysis.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"84 ","pages":"Article 103456"},"PeriodicalIF":3.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146777132","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative evaluation of SNP sequencing workflows for identification of the missing in Vietnam","authors":"Bethany K. Forsythe ,&nbsp;Bas van Haperen ,&nbsp;Lisa Vangeel ,&nbsp;Kieren J. Hill ,&nbsp;Ingo Bastisch ,&nbsp;Phong H. Do ,&nbsp;Thuy T. Hoang ,&nbsp;Lucy Johnson ,&nbsp;Dung T. Le ,&nbsp;Nam N. Nguyen ,&nbsp;Chau Nguyen ,&nbsp;Linh H. Tran ,&nbsp;Vinh V. Tran ,&nbsp;Tuan A. Vu ,&nbsp;Ha H. Chu ,&nbsp;Ha Hoang ,&nbsp;Thanh T. Tran ,&nbsp;Thomas J. Parsons","doi":"10.1016/j.fsigen.2026.103448","DOIUrl":"10.1016/j.fsigen.2026.103448","url":null,"abstract":"<div><div>Hundreds of thousands of remains are still unidentified following the 1955–1975 conflict in Vietnam. DNA identification is increasingly challenging due to extreme degradation of remaining DNA, a large amount of contaminating microbial DNA, and a growing reliance on distant relatives to provide reference samples for comparison. Although mitochondrial DNA is easier to recover than nuclear DNA, by itself it does not have the discriminatory power needed for large-scale identifications. Standard nuclear short tandem repeat (STR) profiling is not an option due to degradation. Instead, massively parallel sequencing (MPS) and its ability to target thousands of single nucleotide polymorphisms (SNPs) must be considered. This paper presents a direct comparison of three targeted SNP sequencing workflows for the identification of highly degraded Vietnamese skeletal remains. Two pre-existing forensic identification mid-density SNP panels were selected for evaluation: the MPSplex panel and the FORCE panel. Both panels are available with library preparation workflows that have the potential of targeting and enriching SNPs from fragmented DNA: QIAseq single primer extension (FORCE and MPSplex panel) and hybridization capture (FORCE panel). Fifteen bone samples from fifteen sets of unidentified Vietnamese skeletal remains of varying quality were processed using three workflows (MPSplex and FORCE using a modified QIAseq protocol and FORCE using hybridization capture) to compare their performance. Family reference genotypes were simulated to estimate the theoretical identification power and performance of the workflows for each sample. All three workflows resulted in SNP profiles that would theoretically support identifications using at least a single first degree relative and up to a single fourth degree relative, from most of the 15 bone samples. The FORCE-Capture workflow had the highest performance based on SNP recovery and theoretical identification power, while FORCE-QIAseq had the lowest. The MPSplex-QIAseq workflow performed similarly to FORCE-Capture with respect to the percentage of targeted SNPs recovered per sample, however, the larger number of SNPs targeted by the FORCE panel appears to outweigh the advantage of the tri-allelic SNPs targeted by the MPSplex-QIAseq workflow. These findings demonstrate that the targeted sequencing of SNPs when combined with optimised, sensitive library preparation techniques, offers a viable path forward in the identification of the missing in Vietnam.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"84 ","pages":"Article 103448"},"PeriodicalIF":3.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147286636","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Contextual DNA samples in evaluations given activity level propositions","authors":"Yoram R. Goedhart ,&nbsp;Jan A. de Koeijer ,&nbsp;Lambertus H.J. Aarts ,&nbsp;Christianne J. de Poot ,&nbsp;Bas Kokshoorn","doi":"10.1016/j.fsigen.2026.103452","DOIUrl":"10.1016/j.fsigen.2026.103452","url":null,"abstract":"<div><div>Recent years have shown a growing effort in the evaluation of DNA given activity level propositions. Yet, its implementation remains challenging for forensic scientists due to the complexity of assessing relevant DNA TPPR mechanisms. One particular challenge is the sparsity of relevant experimental data in general and their applicability in the context of the case at hand. This difficulty is most apparent when considering the presence and quantity of prevalent and background DNA as the variables affecting them are highly specific and case-dependent, complicating their assessment based on literature data alone. It would hence be advantageous to assess prevalent and background DNA using information specific to the case, ideally gathered at the crime scene. We introduce the concept of contextual sampling: the targeted collection of additional samples from the surroundings of crime-related items to assign probabilities on prevalence and background based on case-relevant data. We identify several categories of contextual samples and demonstrate how these can inform sampling strategies and be integrated into Bayesian networks. Our findings show how contextual sampling allows for case-specific probability assignment, thereby reducing dependence on less-representative literature data. We also address practical considerations – including sample number, sampling location, and analytical strategy – and discuss limitations such as resource demands and uncertainties tied to small sample sizes. We conclude that contextual samples can be collected in anticipation of relevant alternative scenarios. This will provide forensic scientists with a valuable tool for casework, offering more nuanced and case-specific evaluations given activity level propositions. Further research could address operational protocols to optimize its implementation in forensic practice.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"84 ","pages":"Article 103452"},"PeriodicalIF":3.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146228695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterizing DNA mixtures with Demixtify","authors":"August E. Woerner ,&nbsp;Benjamin Crysup ,&nbsp;Michael D. Coble","doi":"10.1016/j.fsigen.2026.103451","DOIUrl":"10.1016/j.fsigen.2026.103451","url":null,"abstract":"<div><div>Driven largely by forensic investigative genetic genealogy, whole genome sequencing (WGS) technologies are undergoing rapid adoption by the forensic community. While many tools for interpreting WGS data are readily drawn from the medical genomics communities, software for evaluating DNA mixtures is notably lacking, especially with low-quantity samples. We present Demixtify, a tool for detecting and characterizing DNA mixtures. Genetic analyses were conducted considering a broad range of population groups, coverages and mixture proportions (MPs). These analyses show that estimation of the MP is sensitive to the source population, but mixture detection (down to 0.01×) is less sensitive to these effects. Careful selection of genetic markers can mitigate the effects of population demographics on proportion estimation.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"84 ","pages":"Article 103451"},"PeriodicalIF":3.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146777141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"When DNA leads the way: The introduction of Forensic Investigative Genetic Genealogy in Sweden and its first use in Europe","authors":"Siri Aili Fagerholm ,&nbsp;Ricky Ansell ,&nbsp;Andreas Tillmar","doi":"10.1016/j.fsigen.2026.103432","DOIUrl":"10.1016/j.fsigen.2026.103432","url":null,"abstract":"<div><div>This paper details the first successful use of Forensic Investigative Genetic Genealogy (FIGG) in Europe, which was applied to identify a double murder perpetrator in a 16-year-old unsolved case. By integrating advanced SNP typing with genetic genealogy methods, conclusive leads were generated beyond the reach of conventional forensic approaches. By using this case as an illustrative example, we describe how FIGG can expand the forensic toolkit, reshape investigative as well as forensic strategies, and enable resolution in long-standing unsolved cases. We also reflect on how this case highlights the need to balance technical progress in forensic genetics with legal, social, and ethical aspects. Finally, we outline the journey initiated with this successful pilot case to the structured implementation of FIGG as an operational tool within the Swedish Law Enforcement.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"83 ","pages":"Article 103432"},"PeriodicalIF":3.1,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146049172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Probabilistic genotyping of non-human DNA mixtures: Using MaSTR™ to analyze mixed canine STRs genotyped with DogFiler","authors":"Samantha L. Badgett ,&nbsp;Teresa M. Tiedge ,&nbsp;Taylor B. Parker ,&nbsp;Kim R. Love ,&nbsp;Natalie K. Flack ,&nbsp;Kelly A. Meiklejohn","doi":"10.1016/j.fsigen.2026.103436","DOIUrl":"10.1016/j.fsigen.2026.103436","url":null,"abstract":"<div><div>Individualization in forensic science is commonly achieved by typing short tandem repeats (STRs). Interpretation of resulting electropherograms can be challenging when the input DNA was low quantity, low quality or a mixture of two or more individuals. Probabilistic genotyping (PG) – application of mathematical models and algorithms to analyze mixed STR profiles – tools have been developed to compute a likelihood ratio that an individual is a contributor, and/or deconvolute the mixed profile into single source genotypes. While PG software has been widely implemented in human forensic laboratories, wildlife forensic laboratories have been slow to adopt given a) separate validations would be needed for each STR panel in use, and b) access to and selection of relevant population data needed for analysis could be challenging. This study looked to complete the first evaluation of PG software for the analysis of non-human STR mixtures. We utilized a continuous PG software, MaSTR™ (SoftGenetics), that permits the creation of a model for a non-human STR panel. Artificial mixtures of domestic dog were prepared at varied ratios and genotyped with the published DogFiler STR panel: 1) unrelated two individual (n = 12) and three individual (n = 3) mixtures, with varying degrees of allele sharing, and 2) related individuals (mother and offspring; n = 2) with high allele sharing. For each mixture, true contributor single source profiles were provided to MaSTR™ under varying propositions to assess the impact on the computed likelihood ratio (LR). Additionally for a subset of unrelated two-individual mixtures (n = 6), analyses with true non-contributors were completed to assess false inclusion rates. For replicate analyses (n = 5) on the same mixed profile, the coefficient of variation in the LogLR was 1.83 % and 2.42 %, for two-and three-individual mixtures, respectively. The overall false exclusion rate of true contributors was 0.43 % (LogLR &lt; 6), and the false inclusion rate with true non-contributors was 0 % (LogLR &gt; 6). Despite nearly half of the mixtures tested consisting of individuals with high levels of allele sharing, MaSTR™ was able to complete the analysis and give the expected result (inclusion or exclusion) regardless of the mixture ratio. These results provide promise for use of MaSTR™ (and PG more generally) in wildlife forensic casework. Laboratories interested in implementing PG will need to complete necessary validations and ideally include in testing STR profiles generated from adjudicated case samples, which was outside the scope of this study.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"83 ","pages":"Article 103436"},"PeriodicalIF":3.1,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146081589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Defining key criteria for microhaplotype locus selection in forensic genetics: Progress and recommendations by the Microhaplotype Working Group","authors":"Daniele Podini ,&nbsp;Daniel S. Standage ,&nbsp;Christopher Phillips ,&nbsp;María de la Puente ,&nbsp;Claus Børsting ,&nbsp;Vania Pereira ,&nbsp;Lucinda Davenport ,&nbsp;David Ballard ,&nbsp;Sarah E. Cavanaugh ,&nbsp;Brian Young ,&nbsp;Robert Lagacé ,&nbsp;Kevin M. Kiesler ,&nbsp;Fabio Oldoni ,&nbsp;Pedro Rodriguez ,&nbsp;Chiara Turchi ,&nbsp;Robert A. Bever ,&nbsp;Weibo Liang ,&nbsp;Andrew Pakstis ,&nbsp;Kenneth K. Kidd","doi":"10.1016/j.fsigen.2026.103421","DOIUrl":"10.1016/j.fsigen.2026.103421","url":null,"abstract":"<div><div>The Microhaplotype Working Group (MWG) has made significant progress in defining key criteria for identifying microhaplotype (MH) loci that will enhance forensic genetics. The growing number of publications on MHs reflects increasing interest in these markers and highlights the need for greater standardization in the field. This paper summarizes the group's achievements since its formation following forensic community discussions at the 29th ISFG Congress in 2022. In this paper, we focus on locus nomenclature, allele definitions for MHs, and the challenges of marker identification and characterization. With the progress achieved on the core published MH locus database, MicroHapDB (<span><span>https://github.com/bioforensics/MicroHapDB</span><svg><path></path></svg></span>), it is now possible to use unique names for all currently published MHs. The MWG has reached a consensus on the critical marker characteristics for selecting MH loci for forensic use, with the effective number of alleles (Ae) metric as the primary selection parameter. Criteria for excluding otherwise suitable candidate MHs include genome location, length limitations, close physical linkage with forensic STRs, and sequence complexity considerations. Specifically, MHs in Long and Short Interspersed Nuclear Elements (LINEs and SINEs) and Long Terminal Repeats (LTRs) should be avoided, along with loci longer than ∼250 base pairs (bp). Finally, loci containing Indels or low complexity sequence patterns near their allele-defining SNPs should be excluded. MHs less than roughly 100 bp are potentially useful for typing degraded samples while those up to 250 bp are broadly useful and generally more informative if they include a higher average number of informative SNPs. The potential development of specialized MH panels for applications such as mixture deconvolution or biogeographic ancestry estimation is discussed. Considering these parameters, the MWG has identified 1148 loci theoretically suitable for forensic applications. The assembly of a final panel will maximize Ae while ensuring marker independence and loci with flanking sequence suitable for robust primer designs. This work serves as a foundation for the future selection of a core set of MH loci for forensic applications and development of recommendations for their implementation in forensic casework.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"83 ","pages":"Article 103421"},"PeriodicalIF":3.1,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146081682","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient DNA extraction and sequencing protocol for keratinised materials of animals","authors":"Yongheng Zhou ,&nbsp;Peng Gao ,&nbsp;Liangyu Cui ,&nbsp;Boyang Liu ,&nbsp;Chang Su ,&nbsp;Qi Zhang ,&nbsp;Yue Ma ,&nbsp;Tianming Lan ,&nbsp;Shuhui Yang ,&nbsp;Yanchun Xu","doi":"10.1016/j.fsigen.2026.103437","DOIUrl":"10.1016/j.fsigen.2026.103437","url":null,"abstract":"<div><div>Keratinised tissues represent a valuable non-invasive biological source of mitochondrial and nuclear DNA and hold significant potential for genomic research. However, studies using genomic DNA from keratinised tissues (kmDNA) remain considerably limited, particularly in non-human species, with most studies focusing on human hair. The limited understanding of kmDNA quality constrains its application, particularly in contexts where high-quality DNA is scarce, such as in wildlife conservation and historical research. Thus, this study aimed to develop an efficient DNA extraction and whole-genome sequencing protocol for isolating DNA from diverse keratinised materials. The protocol incorporates several key improvements that enhance the recovery of endogenous DNA from highly degraded samples. This optimised workflow allowed the recovery of DNA fragments as short as 20–30 bp and increased the proportion of endogenous DNA (<em>R</em>_<em>m</em>) by over 20 % on average. The refined library preparation strategy increased sequencing chip utilisation (the ratio of actual output data to its theoretical maximum output) by more than 50 % in some samples. This study provides an optimised sequencing protocol for the systematic evaluation of DNA quality across common keratinised materials, establishing a robust methodological framework for the genetic analysis of complex degraded samples (e.g. historical or field-collected samples). These advancements significantly expand the practical boundaries of molecular biological research under non-ideal conditions.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"83 ","pages":"Article 103437"},"PeriodicalIF":3.1,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146081681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Forensic botany clues: Development of a novel Osmanthus fragrans STR multiplex system","authors":"Kewei Liang ,&nbsp;Pengfei Li ,&nbsp;Wei Zhang ,&nbsp;Chun Shi ,&nbsp;Bin Zhou ,&nbsp;Ting Wu ,&nbsp;Fu Ren ,&nbsp;Fei Guo","doi":"10.1016/j.fsigen.2026.103440","DOIUrl":"10.1016/j.fsigen.2026.103440","url":null,"abstract":"<div><div>Comprehensive research on human DNA genetic markers shows that short tandem repeats (STRs) have found extensive applications in individual identification and kinship testing. Botanical STRs are less commonly employed in forensic science due to the distinctive structure of plant cells and the incomplete state of genome sequencing. This study developed a novel 12-plex system for the individual identification of <em>Osmanthus fragrans</em> (<em>O. fragrans</em>), a species widely distributed across the world's temperate zones, to furnish essential botanical evidence for forensic investigations at crime scenes. Initially, a method for extracting plant DNA from trace samples was improved, which utilized glass beads to break cell walls and yielded significantly higher DNA quantities compared to the traditional method. Subsequently, five novel highly polymorphic STR loci (GHSTR1, GHSTR2, GHSTR4, GHSTR5, and GHSTR6) with trinucleotide repeats were identified from the <em>O. fragrans</em> reference genome (GCA_019395295.1) and characterized (expected heterozygosity &gt; 0.8, polymorphic information content &gt; 0.8, and power of discrimination &gt; 0.9). These loci were integrated with seven previously reported loci (OF5, OF7, OF8, OF9, OF13, OF19, and OSM063) to establish a multiplex system. Developmental validation was conducted in accordance with ISFG and SWGDAM guidelines. The system demonstrated substantial species specificity, high sensitivity (optimal input: 50–500 pg), robust tolerance to common PCR inhibitors (e.g., ≤500 µM hematin and ≤400 ng/µL humic acid), and reliable performance in mixture studies (detecting minor contributors at 1:19–19:1 ratios). PCR conditions were further optimized to 58°C annealing, 20 min elongation, and 28 cycles. The system showed high precision (standard deviation &lt;0.1 bp), accuracy (size deviation within ±0.5 bp), and inter-laboratory concordance. Population analysis of 273 samples revealed the high power of the system effectiveness (combined power of discrimination = 1 − 1.0045 ×10⁻¹⁹ and combined probability of exclusion = 0.993111589879955) despite observed Hardy-Weinberg equilibrium deviations. Ultimately, the system established definitive links between botanical evidence from suspects/victims and primary crime scenes in two real-world cases. In conclusion, the findings of the present study demonstrate the applicability of a novel STR multiplex system for individual identification of <em>O. fragrans</em> trace samples in forensic science. Without these forensic botany clues, these cases may not have been resolved.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"83 ","pages":"Article 103440"},"PeriodicalIF":3.1,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146081590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}