Pub Date : 2025-06-11DOI: 10.1016/j.fsigen.2025.103316
Antonia Heidegger , Martina Unterländer , Lena Ewers , Georg Ausserer Staubmann , Harald Niederstätter , Lisa Marinelli , Angelika Fürst , Jakob Niewöhner , María de la Puente , Ewa Kartasińska , Anna Woźniak , Ewelina Pośpiech , Iris Buckel , Natalie Schneewind , Maja Sidstedt , Marina Ventayol García , François-Xavier Laurent , Ayhan Ulus , Julien Vannier , Anna Delest , Walther Parson
Over the past decade, numerous assays for forensic age estimation based on the analysis of DNA methylation markers have been developed, demonstrating significant potential for use in criminal investigations. Despite these advancements, only few comprehensive evaluation studies were published. In this study, we present findings of an extensive inter-laboratory evaluation of the VISAGE Enhanced Tool and its associated statistical models for epigenetic age estimation in blood and buccal swabs. Six laboratories conducted reproducibility, concordance, and sensitivity assessments using DNA methylation controls alongside blood and saliva samples to evaluate the tool's technical performance. Results demonstrated consistent and reliable DNA methylation quantification across all participating laboratories, with the tool maintaining sensitivity even with a DNA input of 5 ng for bisulfite conversion. To evaluate the age estimation models, 160 blood and 100 buccal swab samples were analysed in three laboratories. The models achieved mean absolute errors (MAEs) of 3.95 years for blood and 4.41 years buccal swabs, which represents an increase of ∼0.7 years for both tissues to the results from the original VISAGE testing set. When comparing results of each laboratory with the original VISAGE testing set, significant differences were found only for age estimation results from blood of one laboratory with an underestimation of chronological age observed within the entire range tested at that laboratory. When excluding this laboratory, the MAE decreased to 3.1 years (N = 89). No significant differences among laboratories were found for buccal swabs. Overall, this study confirms that the VISAGE Enhanced Tool performs robust DNA methylation quantification and reliable age prediction, however protocol and model validation within each laboratory is required upon implementation.
{"title":"Inter-laboratory evaluation of the VISAGE enhanced tool and models for age estimation from blood and buccal cells","authors":"Antonia Heidegger , Martina Unterländer , Lena Ewers , Georg Ausserer Staubmann , Harald Niederstätter , Lisa Marinelli , Angelika Fürst , Jakob Niewöhner , María de la Puente , Ewa Kartasińska , Anna Woźniak , Ewelina Pośpiech , Iris Buckel , Natalie Schneewind , Maja Sidstedt , Marina Ventayol García , François-Xavier Laurent , Ayhan Ulus , Julien Vannier , Anna Delest , Walther Parson","doi":"10.1016/j.fsigen.2025.103316","DOIUrl":"10.1016/j.fsigen.2025.103316","url":null,"abstract":"<div><div>Over the past decade, numerous assays for forensic age estimation based on the analysis of DNA methylation markers have been developed, demonstrating significant potential for use in criminal investigations. Despite these advancements, only few comprehensive evaluation studies were published. In this study, we present findings of an extensive inter-laboratory evaluation of the VISAGE Enhanced Tool and its associated statistical models for epigenetic age estimation in blood and buccal swabs. Six laboratories conducted reproducibility, concordance, and sensitivity assessments using DNA methylation controls alongside blood and saliva samples to evaluate the tool's technical performance. Results demonstrated consistent and reliable DNA methylation quantification across all participating laboratories, with the tool maintaining sensitivity even with a DNA input of 5 ng for bisulfite conversion. To evaluate the age estimation models, 160 blood and 100 buccal swab samples were analysed in three laboratories. The models achieved mean absolute errors (MAEs) of 3.95 years for blood and 4.41 years buccal swabs, which represents an increase of ∼0.7 years for both tissues to the results from the original VISAGE testing set. When comparing results of each laboratory with the original VISAGE testing set, significant differences were found only for age estimation results from blood of one laboratory with an underestimation of chronological age observed within the entire range tested at that laboratory. When excluding this laboratory, the MAE decreased to 3.1 years (N = 89). No significant differences among laboratories were found for buccal swabs. Overall, this study confirms that the VISAGE Enhanced Tool performs robust DNA methylation quantification and reliable age prediction, however protocol and model validation within each laboratory is required upon implementation.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"79 ","pages":"Article 103316"},"PeriodicalIF":3.2,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144298624","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}
Pub Date : 2025-06-01DOI: 10.1016/j.fsigen.2025.103304
Linying Ye , Xiaofeng Zhang , Xueyuan Liu , Litao Huang , Xiaohui Chen , Yangyang Zheng , Jieyu Du , Miaoqiang Lun , Quyi Xu , Weian Du , Chao Liu , Ling Chen
Accurate identification of vaginal fluid is imperative for forensic investigations, particularly in resolving sexual assault cases. Body fluid-specific microbiota are considered potential indicators for identifying body fluids. In this study, we selected vaginal core bacteria based on species level and developed a multiplex PCR system for identifying vaginal fluid in various individual states and mixed stains. This system was validated using a set of 350 samples (245 vaginal samples and 105 other body fluid samples). The system demonstrated high sensitivity, stability against inhibitors, and strong species specificity, showing that saliva and skin swabs were not misidentified as vaginal fluid. Menstrual blood and vaginal fluid exhibited extremely similar identification results. Further validation of individual factors showed that the system was applicable to vaginal samples from female individuals across diverse geographical regions, with varying health statuses and sexual practices. Moreover, the system effectively identified vaginal fluid components in all simulated mixed samples and successfully analyzed aged vaginal samples from actual sexual assault cases. In conclusion, this multiplex PCR system provides a promising tool for the forensic identification of vaginal fluid.
{"title":"A multiplex bacterial assay for identifying vaginal fluid in various individual states and mixture stains","authors":"Linying Ye , Xiaofeng Zhang , Xueyuan Liu , Litao Huang , Xiaohui Chen , Yangyang Zheng , Jieyu Du , Miaoqiang Lun , Quyi Xu , Weian Du , Chao Liu , Ling Chen","doi":"10.1016/j.fsigen.2025.103304","DOIUrl":"10.1016/j.fsigen.2025.103304","url":null,"abstract":"<div><div>Accurate identification of vaginal fluid is imperative for forensic investigations, particularly in resolving sexual assault cases. Body fluid-specific microbiota are considered potential indicators for identifying body fluids. In this study, we selected vaginal core bacteria based on species level and developed a multiplex PCR system for identifying vaginal fluid in various individual states and mixed stains. This system was validated using a set of 350 samples (245 vaginal samples and 105 other body fluid samples). The system demonstrated high sensitivity, stability against inhibitors, and strong species specificity, showing that saliva and skin swabs were not misidentified as vaginal fluid. Menstrual blood and vaginal fluid exhibited extremely similar identification results. Further validation of individual factors showed that the system was applicable to vaginal samples from female individuals across diverse geographical regions, with varying health statuses and sexual practices. Moreover, the system effectively identified vaginal fluid components in all simulated mixed samples and successfully analyzed aged vaginal samples from actual sexual assault cases. In conclusion, this multiplex PCR system provides a promising tool for the forensic identification of vaginal fluid.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"78 ","pages":"Article 103304"},"PeriodicalIF":3.2,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144177756","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}
Pub Date : 2025-05-28DOI: 10.1016/j.fsigen.2025.103307
Mun-Jeong Cho , Seung-Jin Park , Hwan Young Lee , Jong-Lyul Park , Seung Mi Lee , Jae-Yoon Kim , So-Yeon Lee , Seon-Young Kim , Jong Kwan Jun , Soong Deok Lee
In the current forensic environment, short tandem repeat (STR) profiling originating from genomic differences is highly accurate and widely used to identify individuals. However, if the person of interest is a monozygotic (MZ) twin, his or her DNA profile is identical to that of his or her twin; thus, STR profiling cannot discriminate between them. Therefore, DNA methylation, which is known to have different patterns even in MZ twins, has attracted attention as a promising marker to differentiate MZ twins. These epigenetic patterns are affected by environmental factors and age, and distinct DNA methylation patterns have been observed among the three types of MZ twins, i.e., dichorionic-diamniotic, monochorionic-diamniotic, and monochorionic-monoamniotic. To compare DNA methylation profiles among these three types of MZ twins and identify common markers to differentiate MZ twins, we collected cord blood samples from 54 pairs of MZ twins and analyzed their DNA methylation profiles using the Human MethylationEPIC v2.0 platform. The differences in DNA methylation observed among the three types of MZ twins occurred in immune-related regions. Differentially methylated genes identified in both monochorionic-diamniotic and monochorionic-monoamniotic twins were enriched in cytokine signaling and interleukin signaling-related regions. However, differentially methylated genes in dichorionic-diamniotic twins were enriched in PPI at synapse and the neuronal system. To facilitate twin differentiation, we selected a combination of CpG sites that differed between MZ twins and validated this CpG combination in two independent cohorts comprising 118 British MZ twin pairs and 47 Korean MZ twin pairs. Additionally, these selected DNA methylation markers were evaluated in 60 independent samples of MZ twins using pyrosequencing. Our results suggest that the methylation differences observed between MZ twins at birth persist throughout life. Consequently, these CpG site combinations could serve as valuable methylation markers in forensic cases where a suspect is a MZ twin.
{"title":"Genome-wide DNA methylome profiling to differentiate monozygotic twins","authors":"Mun-Jeong Cho , Seung-Jin Park , Hwan Young Lee , Jong-Lyul Park , Seung Mi Lee , Jae-Yoon Kim , So-Yeon Lee , Seon-Young Kim , Jong Kwan Jun , Soong Deok Lee","doi":"10.1016/j.fsigen.2025.103307","DOIUrl":"10.1016/j.fsigen.2025.103307","url":null,"abstract":"<div><div>In the current forensic environment, short tandem repeat (STR) profiling originating from genomic differences is highly accurate and widely used to identify individuals. However, if the person of interest is a monozygotic (MZ) twin, his or her DNA profile is identical to that of his or her twin; thus, STR profiling cannot discriminate between them. Therefore, DNA methylation, which is known to have different patterns even in MZ twins, has attracted attention as a promising marker to differentiate MZ twins. These epigenetic patterns are affected by environmental factors and age, and distinct DNA methylation patterns have been observed among the three types of MZ twins, i.e., dichorionic-diamniotic, monochorionic-diamniotic, and monochorionic-monoamniotic. To compare DNA methylation profiles among these three types of MZ twins and identify common markers to differentiate MZ twins, we collected cord blood samples from 54 pairs of MZ twins and analyzed their DNA methylation profiles using the Human MethylationEPIC v2.0 platform. The differences in DNA methylation observed among the three types of MZ twins occurred in immune-related regions. Differentially methylated genes identified in both monochorionic-diamniotic and monochorionic-monoamniotic twins were enriched in cytokine signaling and interleukin signaling-related regions. However, differentially methylated genes in dichorionic-diamniotic twins were enriched in PPI at synapse and the neuronal system. To facilitate twin differentiation, we selected a combination of CpG sites that differed between MZ twins and validated this CpG combination in two independent cohorts comprising 118 British MZ twin pairs and 47 Korean MZ twin pairs. Additionally, these selected DNA methylation markers were evaluated in 60 independent samples of MZ twins using pyrosequencing. Our results suggest that the methylation differences observed between MZ twins at birth persist throughout life. Consequently, these CpG site combinations could serve as valuable methylation markers in forensic cases where a suspect is a MZ twin.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"79 ","pages":"Article 103307"},"PeriodicalIF":3.2,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144194639","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}
Pub Date : 2025-05-27DOI: 10.1016/j.fsigen.2025.103302
Feriel Ouerghi , Dan E. Krane , Michael D. Edge
Advances in sequencing technology are allowing forensic scientists to access genetic information from increasingly challenging samples. A recently published computational approach, IBDGem, analyzes sequencing reads, including from low-coverage samples, in order to arrive at likelihood ratios for human identification. Here, we show that likelihood ratios produced by IBDGem are best interpreted as testing a null hypothesis different from the traditional one used in a forensic genetics context. In particular, IBDGem tests the hypothesis that the sample comes from an individual who is included in the reference database used to run the method. This null hypothesis is not generally of forensic interest, because the defense hypothesis is not typically that the evidence comes from an individual included in a reference database. Moreover, the computed likelihood ratios can be much larger than likelihood ratios computed for the more standard forensic null hypothesis, often by many orders of magnitude, thus potentially creating an impression of stronger evidence for identity than is warranted. We lay out this result and illustrate it with examples and simulations. As one illustrative example, in a pathological case in which the sequencing error rate is assumed to be zero, if the obtained reads display at least one inconsistency with each member of the reference database, then the likelihood ratio entails a division by zero. We give suggestions for directions that might lead to likelihood ratios that test the typical defense hypothesis.
{"title":"On forensic likelihood ratios from low-coverage sequencing","authors":"Feriel Ouerghi , Dan E. Krane , Michael D. Edge","doi":"10.1016/j.fsigen.2025.103302","DOIUrl":"10.1016/j.fsigen.2025.103302","url":null,"abstract":"<div><div>Advances in sequencing technology are allowing forensic scientists to access genetic information from increasingly challenging samples. A recently published computational approach, <span>IBDGem</span>, analyzes sequencing reads, including from low-coverage samples, in order to arrive at likelihood ratios for human identification. Here, we show that likelihood ratios produced by <span>IBDGem</span> are best interpreted as testing a null hypothesis different from the traditional one used in a forensic genetics context. In particular, <span>IBDGem</span> tests the hypothesis that the sample comes from an individual who is included in the reference database used to run the method. This null hypothesis is not generally of forensic interest, because the defense hypothesis is not typically that the evidence comes from an individual included in a reference database. Moreover, the computed likelihood ratios can be much larger than likelihood ratios computed for the more standard forensic null hypothesis, often by many orders of magnitude, thus potentially creating an impression of stronger evidence for identity than is warranted. We lay out this result and illustrate it with examples and simulations. As one illustrative example, in a pathological case in which the sequencing error rate is assumed to be zero, if the obtained reads display at least one inconsistency with each member of the reference database, then the likelihood ratio entails a division by zero. We give suggestions for directions that might lead to likelihood ratios that test the typical defense hypothesis.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"79 ","pages":"Article 103302"},"PeriodicalIF":3.2,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144221111","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}
Pub Date : 2025-05-27DOI: 10.1016/j.fsigen.2025.103305
Jiarong Zhang , Tingting Yang , Tiantian Shan , Yifan Wei , Qiang Zhu , Ji Zhang , Jiangwei Yan
Nanopore sequencing technology has high portability and affordability of devices, long sequencing reads, and GB-level data yields. In forensic applications, nanopore sequencing has been used in the identification of forensic short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). Comparable to limited STRs, SNP markers have low per-locus discriminating power but are highly sufficient. Although numerous SNP panels consisting of thousands of loci have been developed, till now only ∼100 SNP panels have been evaluated using nanopore sequencing. In this study, we collected 24 DNA samples and employed a 9102 SNPs panel with hybridization capture for enrichment, followed by sequencing using Oxford Nanopore Technologies MinION device and SNP genotyping with NASTRA software. Parallel sequencing and genotyping were performed using Illumina NovaSeq, and we found the consistency of SNP genotypes with nanopore sequencing was over 99.3 %. Next, we filtered X or Y chromosomal markers, 3–6 multiple-allelic SNPs, and further excluded these failing Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD), and obtained 5198 (57.1 %) SNPs. Based on the 5198 SNPs and likelihood ratio methods, 23 pairs of third-degree relatives were discriminated from the unrelated with a 100 % sensitivity and a 98.81 % specificity; 18 pairs of fourth-degree relatives had a 77.78 % sensitivity and 98.47 % specificity. The more close relatives were all correct in the kinship testing.
{"title":"Fourth-degree kinship analysis using 5198 single nucleotide polymorphisms detected by nanopore sequencing platform","authors":"Jiarong Zhang , Tingting Yang , Tiantian Shan , Yifan Wei , Qiang Zhu , Ji Zhang , Jiangwei Yan","doi":"10.1016/j.fsigen.2025.103305","DOIUrl":"10.1016/j.fsigen.2025.103305","url":null,"abstract":"<div><div>Nanopore sequencing technology has high portability and affordability of devices, long sequencing reads, and GB-level data yields. In forensic applications, nanopore sequencing has been used in the identification of forensic short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). Comparable to limited STRs, SNP markers have low per-locus discriminating power but are highly sufficient. Although numerous SNP panels consisting of thousands of loci have been developed, till now only ∼100 SNP panels have been evaluated using nanopore sequencing. In this study, we collected 24 DNA samples and employed a 9102 SNPs panel with hybridization capture for enrichment, followed by sequencing using Oxford Nanopore Technologies MinION device and SNP genotyping with NASTRA software. Parallel sequencing and genotyping were performed using Illumina NovaSeq, and we found the consistency of SNP genotypes with nanopore sequencing was over 99.3 %. Next, we filtered X or Y chromosomal markers, 3–6 multiple-allelic SNPs, and further excluded these failing Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD), and obtained 5198 (57.1 %) SNPs. Based on the 5198 SNPs and likelihood ratio methods, 23 pairs of third-degree relatives were discriminated from the unrelated with a 100 % sensitivity and a 98.81 % specificity; 18 pairs of fourth-degree relatives had a 77.78 % sensitivity and 98.47 % specificity. The more close relatives were all correct in the kinship testing.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"78 ","pages":"Article 103305"},"PeriodicalIF":3.2,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144155088","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}
Pub Date : 2025-05-23DOI: 10.1016/j.fsigen.2025.103303
Nadescha Viviane Hänggi , António Amorim , Heloísa Afonso Costa , Jeppe Dyrberg Andersen , Marie-Louise Kampmann , Cornelius Courts , Maximilian Neis , Denise Syndercombe-Court , Federica Giangasparo , Ane Elida Fonneløp , Helen Johannessen , Thorsten Hadrys , Angelika Fürst , Walther Parson , Harald Niederstätter , Maja Sidstedt , Siri Aili Fagerholm , Titia Sijen , Margreet van den Berge , Erin Hanson , Cordula Haas
Simultaneous identification and association of body fluids to donors can serve as a powerful tool in the criminal investigation of mixed traces. Massively parallel sequencing of mRNA targets not only identifies the origin of the body fluids but may also provide additional contextual information about the body fluid donors of a (binary) mixture using coding region SNPs (cSNPs). Within the European DNA Profiling Group (EDNAP), two consecutive collaborative exercises (3rd and 4th EDNAP exercise) were organized, with the objective to evaluate the performance of two previously published high-resolution mRNA sequencing assays. In the 3rd EDNAP exercise, the BFID-cSNP-BSS assay (cSNPs for blood, saliva, and semen) was evaluated, while in the 4th EDNAP exercise, the BFID-cSNP-6F assay (cSNPs for six fluids/tissues, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin) was tested. Each RNA cSNP assay was accompanied by a genomic DNA assay for the genotyping of the cSNPs in the individual(s) or body fluid donor(s) of interest. A total of 11 laboratories participated in one or both collaborative exercises. In each exercise, the participating laboratories received a set of 16 standardized mock case stains for analysis and were encouraged to analyze additional, self-prepared stains and reference samples. Laboratories could participate using either the Ion Torrent S5™ or the Illumina MiSeq™ sequencing system. The results of the 16 mock case stains were very encouraging in both exercises, as body fluid components could be reliably identified for most of the stains. Since successful donor association depends on the number of body fluid markers covered in the sequencing results, we found that for stains consisting of blood, menstrual blood, vaginal secretion or a mixture thereof, the cSNPs provided substantial genetic discriminatory information for successful association of the respective body fluid to its donor. In mixtures, the difficulty in interpreting the cSNP genotypes might be attributed to the masking effect of the other body fluid(s) present. Body fluid identification and donor association of skin samples proved to be a significant challenge. In conclusion, body fluid identification and donor association using the BFID-cSNP-BSS and -6 F assays is a promising and effective method across laboratories and sequencing platforms.
{"title":"mRNA profiling and donor association of mock casework samples: Results of a 3rd and 4th EDNAP collaborative exercise","authors":"Nadescha Viviane Hänggi , António Amorim , Heloísa Afonso Costa , Jeppe Dyrberg Andersen , Marie-Louise Kampmann , Cornelius Courts , Maximilian Neis , Denise Syndercombe-Court , Federica Giangasparo , Ane Elida Fonneløp , Helen Johannessen , Thorsten Hadrys , Angelika Fürst , Walther Parson , Harald Niederstätter , Maja Sidstedt , Siri Aili Fagerholm , Titia Sijen , Margreet van den Berge , Erin Hanson , Cordula Haas","doi":"10.1016/j.fsigen.2025.103303","DOIUrl":"10.1016/j.fsigen.2025.103303","url":null,"abstract":"<div><div>Simultaneous identification and association of body fluids to donors can serve as a powerful tool in the criminal investigation of mixed traces. Massively parallel sequencing of mRNA targets not only identifies the origin of the body fluids but may also provide additional contextual information about the body fluid donors of a (binary) mixture using coding region SNPs (cSNPs). Within the European DNA Profiling Group (EDNAP), two consecutive collaborative exercises (3rd and 4th EDNAP exercise) were organized, with the objective to evaluate the performance of two previously published high-resolution mRNA sequencing assays. In the 3rd EDNAP exercise, the BFID-cSNP-BSS assay (cSNPs for blood, saliva, and semen) was evaluated, while in the 4th EDNAP exercise, the BFID-cSNP-6F assay (cSNPs for six fluids/tissues, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin) was tested. Each RNA cSNP assay was accompanied by a genomic DNA assay for the genotyping of the cSNPs in the individual(s) or body fluid donor(s) of interest. A total of 11 laboratories participated in one or both collaborative exercises. In each exercise, the participating laboratories received a set of 16 standardized mock case stains for analysis and were encouraged to analyze additional, self-prepared stains and reference samples. Laboratories could participate using either the Ion Torrent S5™ or the Illumina MiSeq™ sequencing system. The results of the 16 mock case stains were very encouraging in both exercises, as body fluid components could be reliably identified for most of the stains. Since successful donor association depends on the number of body fluid markers covered in the sequencing results, we found that for stains consisting of blood, menstrual blood, vaginal secretion or a mixture thereof, the cSNPs provided substantial genetic discriminatory information for successful association of the respective body fluid to its donor. In mixtures, the difficulty in interpreting the cSNP genotypes might be attributed to the masking effect of the other body fluid(s) present. Body fluid identification and donor association of skin samples proved to be a significant challenge. In conclusion, body fluid identification and donor association using the BFID-cSNP-BSS and -6 F assays is a promising and effective method across laboratories and sequencing platforms.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"79 ","pages":"Article 103303"},"PeriodicalIF":3.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144231802","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}
Pub Date : 2025-05-17DOI: 10.1016/j.fsigen.2025.103291
Malte B. Nielsen , Poul S. Eriksen , Helle S. Mogensen , Niels Morling , Mikkel M. Andersen
In genotyping, determining single nucleotide polymorphisms (SNPs) is standard practice, but it becomes difficult when analysing small quantities of input DNA, as is often required in forensic applications. Existing SNP genotyping methods, such as the HID SNP Genotyper Plugin (HSG) from Thermo Fisher Scientific, perform well with adequate DNA input levels but often produce erroneously called genotypes when DNA quantities are low. To mitigate these errors, genotype quality can be checked with the HSG. However, enforcing the HSG’s quality checks decreases the call rate by introducing more no-calls, and it does not eliminate all wrong calls. This study presents and validates a symmetric multinomial logistic regression (SMLR) model designed to enhance genotyping accuracy and call rate with small amounts of DNA. Comprehensive bootstrap and cross-validation analyses across a wide range of DNA quantities demonstrate the robustness and efficiency of the SMLR model in maintaining high call rates without compromising accuracy compared to the HSG. For DNA amounts as low as 31.25 pg, the SMLR method reduced the rate of no-calls by 50.0% relative to the HSG while maintaining the same rate of wrong calls, resulting in a call rate of 96.0%. Similarly, SMLR reduced the rate of wrong calls by 55.6% while maintaining the same call rate, achieving an accuracy of 99.775%. The no-call and wrong-call rates were significantly reduced at 62.5–250 pg DNA. The results highlight the SMLR model’s utility in optimising SNP genotyping at suboptimal DNA concentrations, making it a valuable tool for forensic applications where sample quantity and quality may be decreased. This work reinforces the feasibility of statistical approaches in forensic genotyping and provides a framework for implementing the SMLR method in practical forensic settings. The SMLR model applies to genotyping biallelic data with a signal (e.g. reads, counts, or intensity) for each allele. The model can also improve the allele balance quality check.
{"title":"Enhanced SNP genotyping with symmetric multinomial logistic regression","authors":"Malte B. Nielsen , Poul S. Eriksen , Helle S. Mogensen , Niels Morling , Mikkel M. Andersen","doi":"10.1016/j.fsigen.2025.103291","DOIUrl":"10.1016/j.fsigen.2025.103291","url":null,"abstract":"<div><div>In genotyping, determining single nucleotide polymorphisms (SNPs) is standard practice, but it becomes difficult when analysing small quantities of input DNA, as is often required in forensic applications. Existing SNP genotyping methods, such as the HID SNP Genotyper Plugin (HSG) from Thermo Fisher Scientific, perform well with adequate DNA input levels but often produce erroneously called genotypes when DNA quantities are low. To mitigate these errors, genotype quality can be checked with the HSG. However, enforcing the HSG’s quality checks decreases the call rate by introducing more no-calls, and it does not eliminate all wrong calls. This study presents and validates a symmetric multinomial logistic regression (SMLR) model designed to enhance genotyping accuracy and call rate with small amounts of DNA. Comprehensive bootstrap and cross-validation analyses across a wide range of DNA quantities demonstrate the robustness and efficiency of the SMLR model in maintaining high call rates without compromising accuracy compared to the HSG. For DNA amounts as low as 31.25<!--> <!-->pg, the SMLR method reduced the rate of no-calls by 50.0% relative to the HSG while maintaining the same rate of wrong calls, resulting in a call rate of 96.0%. Similarly, SMLR reduced the rate of wrong calls by 55.6% while maintaining the same call rate, achieving an accuracy of 99.775%. The no-call and wrong-call rates were significantly reduced at 62.5–250<!--> <!-->pg DNA. The results highlight the SMLR model’s utility in optimising SNP genotyping at suboptimal DNA concentrations, making it a valuable tool for forensic applications where sample quantity and quality may be decreased. This work reinforces the feasibility of statistical approaches in forensic genotyping and provides a framework for implementing the SMLR method in practical forensic settings. The SMLR model applies to genotyping biallelic data with a signal (e.g. reads, counts, or intensity) for each allele. The model can also improve the allele balance quality check.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"78 ","pages":"Article 103291"},"PeriodicalIF":3.2,"publicationDate":"2025-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144105510","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}
Pub Date : 2025-05-17DOI: 10.1016/j.fsigen.2025.103296
Quan Xie , Wenting Zhao , Wenhai Liu , Yiru Zhao , Xuanzhu Chen , Jing Li , Zhixiao Fang , Lan Hu , Caixia Li
High-density single nucleotide polymorphism (SNP) genotyping data at varying depths were obtained through whole genome sequencing (WGS). The accuracy of genotyping was evaluated, and methods for forensic SNP genealogy inference using WGS data were explored. The impact of sequencing depth on the accuracy of forensic genealogy inference was also assessed. Samples were sequenced at autosomal depths of 30 × , 14 × , 8 × , and 4 × using the MGISEQ-200RS platform, extracting 645,199 autosomal SNP loci referring the SNP chip panel. After quality control, the Identity by Descent (IBD) algorithm was used to calculate kinship and analyze the biogeographic origin of the samples. The consistency rate of SNP genotyping between sequencing data and SNP chip data exceeded 96.00 %. The IBD algorithm accurately predicted kinship from 1st to 7th degree using autosomal depths of 30 × , 14 × , and 8 × , with one false negative at the 7th degree in 8 × data. The accuracy of SNP genealogy inference from 30 × , 14 × , and 8 × WGS data was not significantly different from that obtained from the SNP chip (p-values: 0.93, 0.83, and 0.54). For 4 × depth data, improvements in quality control and algorithm optimization are needed to enhance genealogy inference accuracy. Additionally, SNP-based biogeographic inference from WGS data were consistent with survey results.
{"title":"Forensic SNP genealogy inference using whole genome sequencing data of varying depths","authors":"Quan Xie , Wenting Zhao , Wenhai Liu , Yiru Zhao , Xuanzhu Chen , Jing Li , Zhixiao Fang , Lan Hu , Caixia Li","doi":"10.1016/j.fsigen.2025.103296","DOIUrl":"10.1016/j.fsigen.2025.103296","url":null,"abstract":"<div><div>High-density single nucleotide polymorphism (SNP) genotyping data at varying depths were obtained through whole genome sequencing (WGS). The accuracy of genotyping was evaluated, and methods for forensic SNP genealogy inference using WGS data were explored. The impact of sequencing depth on the accuracy of forensic genealogy inference was also assessed. Samples were sequenced at autosomal depths of 30 × , 14 × , 8 × , and 4 × using the MGISEQ-200RS platform, extracting 645,199 autosomal SNP loci referring the SNP chip panel. After quality control, the Identity by Descent (IBD) algorithm was used to calculate kinship and analyze the biogeographic origin of the samples. The consistency rate of SNP genotyping between sequencing data and SNP chip data exceeded 96.00 %. The IBD algorithm accurately predicted kinship from 1st to 7th degree using autosomal depths of 30 × , 14 × , and 8 × , with one false negative at the 7th degree in 8 × data. The accuracy of SNP genealogy inference from 30 × , 14 × , and 8 × WGS data was not significantly different from that obtained from the SNP chip (p-values: 0.93, 0.83, and 0.54). For 4 × depth data, improvements in quality control and algorithm optimization are needed to enhance genealogy inference accuracy. Additionally, SNP-based biogeographic inference from WGS data were consistent with survey results.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"79 ","pages":"Article 103296"},"PeriodicalIF":3.2,"publicationDate":"2025-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144195477","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}
Pub Date : 2025-05-13DOI: 10.1016/j.fsigen.2025.103301
Sandra Carbó-Ramírez , Alan Codoñer-Alejos , Mariana Reyes-Prieto , Cristina Bernat , Anabel Gil , Jorge Ruiz-Ramírez , Vicente Soriano-Chirona , Griselda De Marco-Romero , Fernando Gonzalez-Candelas , Llúcia Martinez-Priego
Short tandem repeats (STRs) are widely used in forensic genetics for individual identification. While traditional STR analysis relies on capillary electrophoresis (CE), next-generation sequencing (NGS) offers advantages such as full allelic sequence resolution, improving sensitivity and discrimination power. However, genetic variations in flanking regions can lead to discordant genotyping results between CE and NGS approaches, as well as among different analysis software. During the GEDNAP Proficiency Test 65, a genotyping discrepancy was observed at the D19S433 locus. The sample was analyzed using the ForenSeq® DNA Signature Prep Kit on the MiSeq FGx® Sequencing System, yielding a genotype 11.2,16 when analyzed with the ForenSeq™ Universal Analysis Software. This result differed from the 11.1,16 genotype reported by GEDNAP’s CE-based results. Sequencing data from ForenSeq was further reanalyzed with STRait Razor Online and STRNaming, resulting in a genotype 11.1,16. Additional testing with three different CE kits (AmpFLSTR™ Identifiler™ Plus, NGM SElect™, and GlobalFiler™) produced a 16,16 genotype, leading to three different genotype assignments for the same sample. A 3-bp TCT deletion in the 5’ flanking region of D19S433, located within the International Society for Forensic Genetics (ISFG) minimum reporting range was identified as the cause of these genotyping inconsistencies. Long-read sequencing with PacBio Sequel II technology confirmed that no additional variants were present in the primer binding regions, demonstrating that the TCT deletion alone was responsible for the discrepancies. This study highlights the impact of flanking region mutations on allele calling across different STR typing technologies and the lack of consensus in sequence analysis among bioinformatics pipelines, emphasizing the need to incorporate the ISFG minimum range in the regions sequenced and reported by NGS kits to ensure inter-laboratory and inter-kit consistency, ultimately minimizing discrepancies in forensic STR typing.
{"title":"One sample, three genotypes: A flanking region deletion at the D19S433 locus causes genotyping discrepancies between CE and NGS technologies","authors":"Sandra Carbó-Ramírez , Alan Codoñer-Alejos , Mariana Reyes-Prieto , Cristina Bernat , Anabel Gil , Jorge Ruiz-Ramírez , Vicente Soriano-Chirona , Griselda De Marco-Romero , Fernando Gonzalez-Candelas , Llúcia Martinez-Priego","doi":"10.1016/j.fsigen.2025.103301","DOIUrl":"10.1016/j.fsigen.2025.103301","url":null,"abstract":"<div><div>Short tandem repeats (STRs) are widely used in forensic genetics for individual identification. While traditional STR analysis relies on capillary electrophoresis (CE), next-generation sequencing (NGS) offers advantages such as full allelic sequence resolution, improving sensitivity and discrimination power. However, genetic variations in flanking regions can lead to discordant genotyping results between CE and NGS approaches, as well as among different analysis software. During the GEDNAP Proficiency Test 65, a genotyping discrepancy was observed at the D19S433 locus. The sample was analyzed using the ForenSeq® DNA Signature Prep Kit on the MiSeq FGx® Sequencing System, yielding a genotype 11.2,16 when analyzed with the ForenSeq™ Universal Analysis Software. This result differed from the 11.1,16 genotype reported by GEDNAP’s CE-based results. Sequencing data from ForenSeq was further reanalyzed with STRait Razor Online and STRNaming, resulting in a genotype 11.1,16. Additional testing with three different CE kits (AmpFLSTR™ Identifiler™ Plus, NGM SElect™, and GlobalFiler™) produced a 16,16 genotype, leading to three different genotype assignments for the same sample. A 3-bp TCT deletion in the 5’ flanking region of D19S433, located within the International Society for Forensic Genetics (ISFG) minimum reporting range was identified as the cause of these genotyping inconsistencies. Long-read sequencing with PacBio Sequel II technology confirmed that no additional variants were present in the primer binding regions, demonstrating that the TCT deletion alone was responsible for the discrepancies. This study highlights the impact of flanking region mutations on allele calling across different STR typing technologies and the lack of consensus in sequence analysis among bioinformatics pipelines, emphasizing the need to incorporate the ISFG minimum range in the regions sequenced and reported by NGS kits to ensure inter-laboratory and inter-kit consistency, ultimately minimizing discrepancies in forensic STR typing.</div></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":"78 ","pages":"Article 103301"},"PeriodicalIF":3.2,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144116365","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}
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