Analysis of 26 STRs loci (PowerPlex® Fusion 6C System) in Mestizos from the Mexico City

Analysis of 26 STRs loci (PowerPlex® Fusion 6C System) in Mestizos from the Mexico City | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Analysis of 26 STRs loci (PowerPlex® Fusion 6C System) in Mestizos from the Mexico City Mauro López-Armenta, María Elizbeth Álvarez-Sánchez, Carolina León-Campos, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3856134/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Mar, 2024 Read the published version in Molecular Biology Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Background Short tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes, in order to enable laboratories to use these genetic systems to interpret and solve forensic caseworks. However, in Mexico, there are not studies with the PowerPlex Fusion 6C System, which included 26 STRs (23 autosomal STRs and 3 Y-STRs). Methods and Results A total of 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 321 different alleles were observed, while combined PE and PD were 99.999999809866 and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations. Conclusions The results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied, and justify its possible application in other Mexican Mestizo populations. Forensic genetics STRs PowerPlex Fusion 6C Mexico City 1. Introduction Mestizos (admixed) represents the largest Mexican population (~ 90%), resulting from approximately 500 years of admixture between European, Amerindian and African populations, which arose as a result of the Spanish conquest. While Mexican populations have been extensively studied using classical forensic kits with ~ 15 STRs [ 1 – 3 ], in recent years, new commercial human identification (HID) systems have extended the number of STR loci to enhance forensic casework resolution, which compels the generation of additional population studies. In the case of Mestizo populations, this task is crucial due to their population structure, which stems from the contrasting ancestral gradient observed throughout the Mexican territory: European ancestry increases to the Northwest, whereas the Amerindian enlarges to the Southeast region [ 4 – 6 ]. Some studies have reported Mexican population data based on kits with ≥ 20 STRs loci, such as the 20 STRs of the PowerPlex 21 [ 6 ], the 22 STRs of the GlobalFiler kit [ 7 , 8 ], and the 23 STRs of the PowerPlex Fusion [ 9 ]. Nevertheless, there are no currently studies available in Mexican populations employing the PowerPlex Fusion 6C System. This kit includes 27 genetic markers (26 STRs and the sex-determining AMEL marker). Therefore, the objective of this study was to analyze the 26 STRs of the PowerPlex Fusion 6C System to obtain statistical parameters of forensic interest and explore the genetic structure of the Mexico City population, which serves as the capital of Mexico and is the main economic and cultural Metropolis of the country. 2. Materials and Methods Population sample A total of 600 samples from Mexico City were included in this study (233 females and 367 males), comprising both living persons (LP, n = 300) and human cadavers (HC, n = 300). LP samples were collected in the de Laboratory of Genetics of the Instituto de Servicios Periciales y Ciencias Forenses (INCIFO), and all volunteers provided informed consent according to the Helsinki Declaration. HC samples were cataloged as missing persons and were discovered in various boroughs of the Mexico City, as following: Álvaro Obregón (LP n = 23, HC n = 27), Azcapotzalco (LP n = 13, HC n = 15), Benito Juárez (LP n = 14, HC n = 14), Coyoacán (LP n = 20, HC n = 20), Cuajimalpa de Morelos (LP n = 7, HC n = 7), Cuauhtémoc (LP n = 18, HC n = 18), Gustavo A. Madero (LP n = 38, HC n = 38), Iztacalco (LP n = 12, HC n = 14), Iztapalapa (LP n = 56, HC n = 64), La Magdalena Contreras (LP n = 8, HC n = 8), Miguel Hidalgo (LP n = 12, HC n = 16), Milpa Alta (LP n = 4, HC n = 6), Tláhuac (LP n = 11, HC n = 15), Tlalpan (LP n = 21, HC n = 25), Venustiano Carranza (LP n = 10, HC n = 18) and Xochimilco (LP n = 11, HC n = 17). HC were genotyped for human identification purposes as part of the casework at INCIFO. In all cases, a negative identification was reported with any LP included in this work, confirming that all individuals are unrelated. This research project received approval from the Comité de Ética en Investigación Forense CEI-INCIFO (CEI-015/019). DNA extraction Samples from LP were collected from oral mucosa using a swab, which was air-dried for preservation before use. Half of the swab was lysed in 400 µl of QG buffer, with the addition of 10 µl of proteinase K at a concentration of 10 mg/ml. The samples were then incubated at 56°C for 15 minutes. The resulting supernatant was processed through a QIAquick spin column (Qiagen, Hilden, Germany) according to the manufacturer's instructions. HC tissue samples were obtained from the cartilaginous tissue of rib arches, with removal of the perichondrium. The cartilage was cleaned using a commercial detergent, agitated, and rinsed with water. Subsequently, it was dehydrated in a desiccator. The clean and dry cartilage was powdered using a rotary tool and sterile metal grinding bits. Thirty milligrams of cartilage powder were then mixed with 250 µl of digestion buffer at pH 8 (10mM Tris, 25mM EDTA, 10mM NaCl, 0.5% SDS). To each sample, 4 µl of 1M DTT and 10 µl of proteinase K at a concentration of 10 mg/ml were added. The samples were incubated at 56°C overnight and then purified using QIAquick spin columns (Qiagen, Hilden, Germany) following the manufacturer's instructions. The extracted DNA was quantified using the Investigator Quantiplex kit (Qiagen, Hilden, Germany) on a 7500 Real-Time PCR System (Applied Biosystems), following the manufacturer's instructions. STR genotyping Amplification of 23 autosomal STRs (D3S1358, D1S1656, D2S441, D10S1248, D13S317, PENTA E, D16S539, D18S51, D2S1338, CSF1PO, PENTA D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045, and SE33), along with three Y-linked STRs (DYS391, DYS576, and DYS570) and Amelogenin, was conducted using the PowerPlex Fusion 6C System following the manufacturer's instructions. The amplified fragments were separated by capillary electrophoresis on a 3500 Genetic Analyzer (Applied Biosystems). Genotype analysis was performed using GeneMapper ID-X v1.5 software (Thermo Fisher Scientific). Positive and negative controls were included in all experimental runs. Statistical analysis This study follows the guidelines and recommendations suggested for announcement of population data [ 10 ]. The following statistical parameters of forensic interest were estimated with the STRAF 2.0.8 software [ 11 ]: Power of discrimination (PD), power of exclusion (PE), polymorphism information content (PIC), typical paternity index (TPI), genetic diversity (GD) and heterozygosity (H). Minimum allele frequencies (MAF) were computed according to NRC recommendations as 5/2N [ 12 ]. For the Y-linked loci, the following parameters were calculated: a) haplotype frequencies, calculated by direct counting; b) discriminatory capacity (DC), estimated by dividing the number of different haplotypes by the number of total haplotypes; c) the theoretical heterozygosity, estimated as follows: He = 1 − ∑pi2, where pi represents the frequency of the allele i. Hardy–Weinberg expectations (HWE) by locus and linkage disequilibrium (LD) between pair of loci by means of exact tests were calculated with the Genetic Data Analysis (GDA) v.1.1 software [ 13 ]. AMOVA test, Fst genetic distances and pairwise comparison (Fst p values) were computed with the Arlequin v3.5.1.2 software [ 14 ]. Genetic distances were graphically represented in a multidimensional scaling (MDS) plot with the program SPSS v.19 for Windows [ 15 ]. For the interpopulation analysis, the following previously published populations were included as reference: African, Caucasian, and Hispanic populations from USA [ 16 ], as well as Mestizo populations from Jalisco (West) [ 9 ] and Monterrey (state of Nuevo León, Northeast) [ 7 ]. For compatibility purposes, the interpopulation analyses were conducted with 22 STRs. 3. Results and discussion Forensic parameters The complete STR genotype dataset of the Mestizo population from the Mexico City is available in Table S1 . Allele frequencies, forensic parameters and HWE values are shown in Table 1 . For autosomal STRs, 321 different alleles were observed, which represents more than 20% additional alleles compared to those reported for the Mestizo population from the West region of Mexico [ 9 ]. This result emphasizes the necessity of analyzing large genotype population databases to detect low-frequency alleles (rare alleles) and to more accurately represent the populations under examination, as demonstrated in this paper. The estimated allelic frequencies and statistical parameters of forensic interest are presented in Table 1 . The SE33 locus exhibited the highest allele diversity (n = 44), while D3S1358 and D5S818 displayed the lower number of different alleles (n = 8). For autosomal loci, SE33 demonstrated the highest values for PD (0.9911), PE (0.8502), GD (0.9379) and PIC (0.9335), whereas D22S1045 showed the lowest PD (0.8002) and PE (0.2652) values (Table 1 ). Meanwhile, the most informative Y-STR was DYS570, with a Het, PD and PE of 0.7967, whereas DYS391 showed the lower values (0.5402). A discriminatory capacity of 27.04% was observed (99 different haplotypes) (Table S2 ), and although that value is considerably lower than those obtained in currently employed Y-STR kits [ 17 , 18 ], it must be noted that this difference is attributed to the variance in the number of loci. Nevertheless, this data is crucial for enhancing the interpretation and results of forensic casework, as Y-STR haplotype frequencies can be integrated into the autosomal STR calculations. The combined PD for autosomal STRs was 99.99999999999999999999999818795% (1-[1.8 X 10 − 27 ]), and the combined PE was 99.999999809866% (1-[1.9X10 − 09 ]), which are considerably higher than those observed previously in Mexican populations based on 20 and 22 STRs [ 6 , 7 , 9 ]. After Bonferroni correction (0.05/23 = 0.0022), all STR markers were in line with the Hardy-Weinberg equilibrium expectations, except for Penta E (p = 0.000, Table 1 ). LD tests displayed no significant association between pair of loci, after Bonferroni correction. Genetic distances Genetic distances and p-values are provided in Table S3 . Mexican populations exhibited low but significant genetic distances among them. As anticipated, they displayed higher genetic distances from the USA populations. Interestingly, the West region of Mexico is the closest to the European population, suggesting the most elevated European ancestry. This trend is followed by Monterrey City, and lastly, by Mexico City (Figure S1 ). These findings align with the previously observed pattern of ancestry gradient in Mexican populations [ 4 – 6 ]. Population structure The AMOVA analysis indicated low variation between Mexican-Mestizo and the major ethnic groups from the USA (Fst = 0.0133, p = 0.0000). However, when populations were stratified into two groups (Mexican versus USA populations), the differentiation among the groups nearly doubled (Fst = 0.0217, p = 0.0000). Conversely, when the three Mexican-Mestizo populations were consolidated into one group, the differentiation among populations decreased by approximately eighteen times (Fst = 0.0012, p = 0.0000). Putting together, these findings underscore the importance of scrutinizing forensic parameters and allele frequencies in populations exhibiting substantial genetic differentiation, such as the Mexican and USA populations. On the other hand, the low levels of differentiation in Mexican-Mestizo observed, both in genetic distances and AMOVA test, suggest the possibility of using forensic data generated from Mexico City to resolve human identification casework in other Mexican populations where databases are not available. 4. Conclusions To the best of our knowledge, this represents the first report utilizing the 26 STRs encompassed in the PowerPlex Fusion 6C System in Mexican populations, extending to any Mestizo population in the Americas. The allele diversity, as well as the values of combined PE and PD reported in this work, represents a considerable increase compared to previous studies with fewer STR loci. This allows employing the results reported herein for human identification purposes. Furthermore, the low and significant differentiation between Mexican populations justifies the data reported herein for implementation in solving casework, even in other Mexican Mestizo populations. This work signifies an effort to generate large forensic databases to better represent the Mexican population with the new kits that include increased STR loci. Declarations Ethical approach This research project received approval from the Comité de Ética en Investigación Forense CEI-INCIFO (CEI-015/019). All live volunteers provided an informed consent according the Helsinki declaration. Samples from human cadavers were used with a firm commitment to ensure respect for and adherence to ethical principles at every stage of the research. Rigorous ethical protocols were followed to ensure the confidentiality of the information and proper handling of the samples, respecting at all times the dignity and fundamental rights of the individual donors. This ethical approach not only constitutes a moral imperative but also strengthens the integrity and validity of the results obtained, thus contributing to the responsible advancement of scientific knowledge in the forensic field. Declarations of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by M L-A, C L-C, BZ L-S and AD S-M. The infrastructure, materials and equipment were provided by M L-A, ME A-S, H R-V and JF M-V. The first draft of the manuscript was written by M L-A, ME A-S and JA A-V. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability Statement The complete genotype dataset, haplotype frequencies and genetic distances associated with this article can be found as Supplementary data in the online version. References Luna-Vazquez A, Vilchis-Dorantes G, Aguilar-Ruiz MO, Bautista-Rivas A, Rojo-Nava AL, Rios-Barrios E, Rangel-Villalobos H (2005) Population data for 15 loci (Identifiler Kit) in a sample from the Valley of Mexico. 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Armonk, NY Hill CR, Duewer DL, Kline MC, Coble MD et al (2013) U.S. population data for 29 autosomal STR loci. Forensic Sci International: Genet 7(4):e82–e83. 10.1016/j.fsigen.2012.12.004 López-Ramírez YL, Aguilar-Velázquez JA, López-Armenta M, Ruiz-Hernández M, Rangel-Villalobos H (2019) Paternal lineages and forensic parameters based on 23 Y-STRs (Powerplex® Y23) in Mestizo males from Mexico City. Int J Legal Med 133(2):551–553 Ramos-González B, Aguilar-Velázquez JA, Chávez-Briones MDL, Escareño-Hernández MDR, Alfaro-Lopez E, Rangel-Villalobos H (2017) Genetic population data of three Y-STR genetic systems in Mexican-Mestizos from Monterrey, Nuevo León (Northeast, Mexico). Forensic Sci International: Genet 29:e21–e22 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx SupplementaryFigureS1.tif SupplementaryTableS1.xlsx SupplementaryTableS2.xlsx SupplementaryTableS3.xlsx Cite Share Download PDF Status: Published Journal Publication published 22 Mar, 2024 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 05 Feb, 2024 Reviews received at journal 05 Feb, 2024 Reviewers agreed at journal 17 Jan, 2024 Reviewers invited by journal 15 Jan, 2024 Submission checks completed at journal 13 Jan, 2024 Editor assigned by journal 13 Jan, 2024 First submitted to journal 12 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Introduction","content":"\u003cp\u003eMestizos (admixed) represents the largest Mexican population (~\u0026thinsp;90%), resulting from approximately 500 years of admixture between European, Amerindian and African populations, which arose as a result of the Spanish conquest. While Mexican populations have been extensively studied using classical forensic kits with ~\u0026thinsp;15 STRs [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], in recent years, new commercial human identification (HID) systems have extended the number of STR loci to enhance forensic casework resolution, which compels the generation of additional population studies. In the case of Mestizo populations, this task is crucial due to their population structure, which stems from the contrasting ancestral gradient observed throughout the Mexican territory: European ancestry increases to the Northwest, whereas the Amerindian enlarges to the Southeast region [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome studies have reported Mexican population data based on kits with \u0026ge;\u0026thinsp;20 STRs loci, such as the 20 STRs of the PowerPlex 21 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], the 22 STRs of the GlobalFiler kit [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and the 23 STRs of the PowerPlex Fusion [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nevertheless, there are no currently studies available in Mexican populations employing the PowerPlex Fusion 6C System. This kit includes 27 genetic markers (26 STRs and the sex-determining AMEL marker). Therefore, the objective of this study was to analyze the 26 STRs of the PowerPlex Fusion 6C System to obtain statistical parameters of forensic interest and explore the genetic structure of the Mexico City population, which serves as the capital of Mexico and is the main economic and cultural Metropolis of the country.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cb\u003ePopulation sample\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA total of 600 samples from Mexico City were included in this study (233 females and 367 males), comprising both living persons (LP, n\u0026thinsp;=\u0026thinsp;300) and human cadavers (HC, n\u0026thinsp;=\u0026thinsp;300). LP samples were collected in the de Laboratory of Genetics of the Instituto de Servicios Periciales y Ciencias Forenses (INCIFO), and all volunteers provided informed consent according to the Helsinki Declaration. HC samples were cataloged as missing persons and were discovered in various boroughs of the Mexico City, as following: \u0026Aacute;lvaro Obreg\u0026oacute;n (LP n\u0026thinsp;=\u0026thinsp;23, HC n\u0026thinsp;=\u0026thinsp;27), Azcapotzalco (LP n\u0026thinsp;=\u0026thinsp;13, HC n\u0026thinsp;=\u0026thinsp;15), Benito Ju\u0026aacute;rez (LP n\u0026thinsp;=\u0026thinsp;14, HC n\u0026thinsp;=\u0026thinsp;14), Coyoac\u0026aacute;n (LP n\u0026thinsp;=\u0026thinsp;20, HC n\u0026thinsp;=\u0026thinsp;20), Cuajimalpa de Morelos (LP n\u0026thinsp;=\u0026thinsp;7, HC n\u0026thinsp;=\u0026thinsp;7), Cuauht\u0026eacute;moc (LP n\u0026thinsp;=\u0026thinsp;18, HC n\u0026thinsp;=\u0026thinsp;18), Gustavo A. Madero (LP n\u0026thinsp;=\u0026thinsp;38, HC n\u0026thinsp;=\u0026thinsp;38), Iztacalco (LP n\u0026thinsp;=\u0026thinsp;12, HC n\u0026thinsp;=\u0026thinsp;14), Iztapalapa (LP n\u0026thinsp;=\u0026thinsp;56, HC n\u0026thinsp;=\u0026thinsp;64), La Magdalena Contreras (LP n\u0026thinsp;=\u0026thinsp;8, HC n\u0026thinsp;=\u0026thinsp;8), Miguel Hidalgo (LP n\u0026thinsp;=\u0026thinsp;12, HC n\u0026thinsp;=\u0026thinsp;16), Milpa Alta (LP n\u0026thinsp;=\u0026thinsp;4, HC n\u0026thinsp;=\u0026thinsp;6), Tl\u0026aacute;huac (LP n\u0026thinsp;=\u0026thinsp;11, HC n\u0026thinsp;=\u0026thinsp;15), Tlalpan (LP n\u0026thinsp;=\u0026thinsp;21, HC n\u0026thinsp;=\u0026thinsp;25), Venustiano Carranza (LP n\u0026thinsp;=\u0026thinsp;10, HC n\u0026thinsp;=\u0026thinsp;18) and Xochimilco (LP n\u0026thinsp;=\u0026thinsp;11, HC n\u0026thinsp;=\u0026thinsp;17). HC were genotyped for human identification purposes as part of the casework at INCIFO. In all cases, a negative identification was reported with any LP included in this work, confirming that all individuals are unrelated. This research project received approval from the Comit\u0026eacute; de \u0026Eacute;tica en Investigaci\u0026oacute;n Forense CEI-INCIFO (CEI-015/019).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA extraction\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSamples from LP were collected from oral mucosa using a swab, which was air-dried for preservation before use. Half of the swab was lysed in 400 \u0026micro;l of QG buffer, with the addition of 10 \u0026micro;l of proteinase K at a concentration of 10 mg/ml. The samples were then incubated at 56\u0026deg;C for 15 minutes. The resulting supernatant was processed through a QIAquick spin column (Qiagen, Hilden, Germany) according to the manufacturer's instructions. HC tissue samples were obtained from the cartilaginous tissue of rib arches, with removal of the perichondrium. The cartilage was cleaned using a commercial detergent, agitated, and rinsed with water. Subsequently, it was dehydrated in a desiccator. The clean and dry cartilage was powdered using a rotary tool and sterile metal grinding bits. Thirty milligrams of cartilage powder were then mixed with 250 \u0026micro;l of digestion buffer at pH 8 (10mM Tris, 25mM EDTA, 10mM NaCl, 0.5% SDS). To each sample, 4 \u0026micro;l of 1M DTT and 10 \u0026micro;l of proteinase K at a concentration of 10 mg/ml were added. The samples were incubated at 56\u0026deg;C overnight and then purified using QIAquick spin columns (Qiagen, Hilden, Germany) following the manufacturer's instructions. The extracted DNA was quantified using the Investigator Quantiplex kit (Qiagen, Hilden, Germany) on a 7500 Real-Time PCR System (Applied Biosystems), following the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSTR genotyping\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAmplification of 23 autosomal STRs (D3S1358, D1S1656, D2S441, D10S1248, D13S317, PENTA E, D16S539, D18S51, D2S1338, CSF1PO, PENTA D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045, and SE33), along with three Y-linked STRs (DYS391, DYS576, and DYS570) and Amelogenin, was conducted using the PowerPlex Fusion 6C System following the manufacturer's instructions. The amplified fragments were separated by capillary electrophoresis on a 3500 Genetic Analyzer (Applied Biosystems). Genotype analysis was performed using GeneMapper ID-X v1.5 software (Thermo Fisher Scientific). Positive and negative controls were included in all experimental runs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study follows the guidelines and recommendations suggested for announcement of population data [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The following statistical parameters of forensic interest were estimated with the STRAF 2.0.8 software [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]: Power of discrimination (PD), power of exclusion (PE), polymorphism information content (PIC), typical paternity index (TPI), genetic diversity (GD) and heterozygosity (H). Minimum allele frequencies (MAF) were computed according to NRC recommendations as 5/2N [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For the Y-linked loci, the following parameters were calculated: a) haplotype frequencies, calculated by direct counting; b) discriminatory capacity (DC), estimated by dividing the number of different haplotypes by the number of total haplotypes; c) the theoretical heterozygosity, estimated as follows: He\u0026thinsp;=\u0026thinsp;1 \u0026minus; \u0026sum;pi2, where pi represents the frequency of the allele i. Hardy\u0026ndash;Weinberg expectations (HWE) by locus and linkage disequilibrium (LD) between pair of loci by means of exact tests were calculated with the Genetic Data Analysis (GDA) v.1.1 software [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. AMOVA test, Fst genetic distances and pairwise comparison (Fst p values) were computed with the Arlequin v3.5.1.2 software [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Genetic distances were graphically represented in a multidimensional scaling (MDS) plot with the program SPSS v.19 for Windows [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For the interpopulation analysis, the following previously published populations were included as reference: African, Caucasian, and Hispanic populations from USA [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], as well as Mestizo populations from Jalisco (West) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and Monterrey (state of Nuevo Le\u0026oacute;n, Northeast) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For compatibility purposes, the interpopulation analyses were conducted with 22 STRs.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eForensic parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete STR genotype dataset of the Mestizo population from the Mexico City is available in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. Allele frequencies, forensic parameters and HWE values are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. For autosomal STRs, 321 different alleles were observed, which represents more than 20% additional alleles compared to those reported for the Mestizo population from the West region of Mexico [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. This result emphasizes the necessity of analyzing large genotype population databases to detect low-frequency alleles (rare alleles) and to more accurately represent the populations under examination, as demonstrated in this paper. The estimated allelic frequencies and statistical parameters of forensic interest are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The SE33 locus exhibited the highest allele diversity (n\u0026thinsp;=\u0026thinsp;44), while D3S1358 and D5S818 displayed the lower number of different alleles (n\u0026thinsp;=\u0026thinsp;8). For autosomal loci, SE33 demonstrated the highest values for PD (0.9911), PE (0.8502), GD (0.9379) and PIC (0.9335), whereas D22S1045 showed the lowest PD (0.8002) and PE (0.2652) values (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, the most informative Y-STR was DYS570, with a Het, PD and PE of 0.7967, whereas DYS391 showed the lower values (0.5402). A discriminatory capacity of 27.04% was observed (99 different haplotypes) (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e), and although that value is considerably lower than those obtained in currently employed Y-STR kits [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], it must be noted that this difference is attributed to the variance in the number of loci. Nevertheless, this data is crucial for enhancing the interpretation and results of forensic casework, as Y-STR haplotype frequencies can be integrated into the autosomal STR calculations.\u003c/p\u003e\n\u003cp\u003eThe combined PD for autosomal STRs was 99.99999999999999999999999818795% (1-[1.8 X 10\u003csup\u003e\u0026minus;\u0026thinsp;27\u003c/sup\u003e]), and the combined PE was 99.999999809866% (1-[1.9X10\u003csup\u003e\u0026minus;\u0026thinsp;09\u003c/sup\u003e]), which are considerably higher than those observed previously in Mexican populations based on 20 and 22 STRs [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. After Bonferroni correction (0.05/23\u0026thinsp;=\u0026thinsp;0.0022), all STR markers were in line with the Hardy-Weinberg equilibrium expectations, except for Penta E (p\u0026thinsp;=\u0026thinsp;0.000, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). LD tests displayed no significant association between pair of loci, after Bonferroni correction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic distances\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenetic distances and p-values are provided in Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e. Mexican populations exhibited low but significant genetic distances among them. As anticipated, they displayed higher genetic distances from the USA populations. Interestingly, the West region of Mexico is the closest to the European population, suggesting the most elevated European ancestry. This trend is followed by Monterrey City, and lastly, by Mexico City (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). These findings align with the previously observed pattern of ancestry gradient in Mexican populations [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePopulation structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AMOVA analysis indicated low variation between Mexican-Mestizo and the major ethnic groups from the USA (Fst\u0026thinsp;=\u0026thinsp;0.0133, p\u0026thinsp;=\u0026thinsp;0.0000). However, when populations were stratified into two groups (Mexican \u003cem\u003eversus\u003c/em\u003e USA populations), the differentiation among the groups nearly doubled (Fst\u0026thinsp;=\u0026thinsp;0.0217, p\u0026thinsp;=\u0026thinsp;0.0000). Conversely, when the three Mexican-Mestizo populations were consolidated into one group, the differentiation among populations decreased by approximately eighteen times (Fst\u0026thinsp;=\u0026thinsp;0.0012, p\u0026thinsp;=\u0026thinsp;0.0000). Putting together, these findings underscore the importance of scrutinizing forensic parameters and allele frequencies in populations exhibiting substantial genetic differentiation, such as the Mexican and USA populations. On the other hand, the low levels of differentiation in Mexican-Mestizo observed, both in genetic distances and AMOVA test, suggest the possibility of using forensic data generated from Mexico City to resolve human identification casework in other Mexican populations where databases are not available.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eTo the best of our knowledge, this represents the first report utilizing the 26 STRs encompassed in the PowerPlex Fusion 6C System in Mexican populations, extending to any Mestizo population in the Americas. The allele diversity, as well as the values of combined PE and PD reported in this work, represents a considerable increase compared to previous studies with fewer STR loci. This allows employing the results reported herein for human identification purposes. Furthermore, the low and significant differentiation between Mexican populations justifies the data reported herein for implementation in solving casework, even in other Mexican Mestizo populations. This work signifies an effort to generate large forensic databases to better represent the Mexican population with the new kits that include increased STR loci.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eEthical approach\u003c/h2\u003e\n\u003cp\u003eThis research project received approval from the Comit\u0026eacute; de \u0026Eacute;tica en Investigaci\u0026oacute;n Forense CEI-INCIFO (CEI-015/019). All live volunteers provided an informed consent according the Helsinki declaration. Samples from human cadavers were used with a firm commitment to ensure respect for and adherence to ethical principles at every stage of the research. Rigorous ethical protocols were followed to ensure the confidentiality of the information and proper handling of the samples, respecting at all times the dignity and fundamental rights of the individual donors. This ethical approach not only constitutes a moral imperative but also strengthens the integrity and validity of the results obtained, thus contributing to the responsible advancement of scientific knowledge in the forensic field.\u003c/p\u003e\n\u003ch2\u003eDeclarations of competing interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by M L-A, C L-C, BZ L-S and AD S-M. The infrastructure, materials and equipment were provided by M L-A, ME A-S, H R-V and JF M-V. The first draft of the manuscript was written by M L-A, ME A-S and JA A-V. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eThe complete genotype dataset, haplotype frequencies and genetic distances associated with this article can be found as Supplementary data in the online version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLuna-Vazquez A, Vilchis-Dorantes G, Aguilar-Ruiz MO, Bautista-Rivas A, Rojo-Nava AL, Rios-Barrios E, Rangel-Villalobos H (2005) Population data for 15 loci (Identifiler Kit) in a sample from the Valley of Mexico. 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Forensic Sci International: Genet 29:e21\u0026ndash;e22\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Forensic genetics, STRs, PowerPlex Fusion 6C, Mexico City","lastPublishedDoi":"10.21203/rs.3.rs-3856134/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3856134/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eShort tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes, in order to enable laboratories to use these genetic systems to interpret and solve forensic caseworks. However, in Mexico, there are not studies with the PowerPlex Fusion 6C System, which included 26 STRs (23 autosomal STRs and 3 Y-STRs).\u003c/p\u003e\u003ch2\u003eMethods and Results\u003c/h2\u003e \u003cp\u003eA total of 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 321 different alleles were observed, while combined PE and PD were 99.999999809866 and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied, and justify its possible application in other Mexican Mestizo populations.\u003c/p\u003e","manuscriptTitle":"Analysis of 26 STRs loci (PowerPlex® Fusion 6C System) in Mestizos from the Mexico City","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 09:57:31","doi":"10.21203/rs.3.rs-3856134/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-05T10:28:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-05T09:27:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0fb75fe3-d447-4eee-bdaf-714469fe9197","date":"2024-01-17T14:25:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-15T13:42:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-13T15:25:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-13T15:25:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2024-01-12T07:38:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"beef8409-f943-423f-a12f-3f6ccebd90da","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-25T15:13:11+00:00","versionOfRecord":{"articleIdentity":"rs-3856134","link":"https://doi.org/10.1007/s11033-024-09390-z","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2024-03-22 15:01:14","publishedOnDateReadable":"March 22nd, 2024"},"versionCreatedAt":"2024-01-16 09:57:31","video":"","vorDoi":"10.1007/s11033-024-09390-z","vorDoiUrl":"https://doi.org/10.1007/s11033-024-09390-z","workflowStages":[]},"version":"v1","identity":"rs-3856134","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3856134","identity":"rs-3856134","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}