Associations between forensic loci and expression levels of neighboring genes may compromise medical privacy.

A set of 20 short tandem repeats (STRs) is used by the US criminal justice system to identify suspects and to maintain a database of genetic profiles for individuals who have been previously convicted or arrested. Some of these STRs were identified in the 1990s, with a preference for markers in putative gene deserts to avoid forensic profiles revealing protected medical information. We revisit that assumption, investigating whether forensic genetic profiles reveal information about gene-expression variation or potential medical information. We find six significant correlations (false discovery rate = 0.23) between the forensic STRs and the expression levels of neighboring genes in lymphoblastoid cell lines. We explore possible mechanisms for these associations, showing evidence compatible with forensic STRs causing expression variation or being in linkage disequilibrium with a causal locus in three cases and weaker or potentially spurious associations in the other three cases. Together, these results suggest that forensic genetic loci may reveal expression levels and, perhaps, medical information.

Developmental Validation of the Microreader 23HS Plex ID System: A Novel Supplementary Non-CODIS STR Multiplex Assay for Forensic Application.

A novel supplementary non-CODIS STR multiplex assay designated as the "Microreader 23HS Plex ID System" was developed. The Microreader 23HS Plex ID System enables simultaneous profiling of 23 STR loci and the amelogenin locus. The majority of these loci are non-CODIS STRs (D4S2408, D9S2157, D20S161, D3S2459, D18S1364, D13S305, D1S2142, D19S400, D6S1017, D7S1517, D2S1776, D2S1360, D3S1744, D16S3391, D3S1545, D11S4463, D20S85, D1S549, D10S2325, D21S2055), with the exception of three CODIS STRs (D2S441, D12S391, and D22S1045). Followed the recommendations of Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese validation standards, a comprehensive set of validation studies were conducted, encompassing PCR conditions, stutter ratio and peak height balance, sensitivity, precision and accuracy, reproducibility, species specificity, inhibition, as well as mixture testing. The results demonstrated that the Microreader 23HS Plex ID System is a reliable and robust assay, with well-balanced peak heights, high precision and accuracy, species specificity, and resistance to common inhibitors. The sensitivity of the assay was determined to be 0.125 ng of template DNA. In mixture study, all minor alleles were detected in two-sample mixtures across various ratios (1:19, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 4:1, 9:1, and 19:1). In population study, a total of 500 unrelated individuals of Han ethnicity from East China were genotyped. The allele frequencies and forensic population genetic parameters were calculated, with a cumulative random match probability of 7.757 × 10-27, and a total power of discrimination exceeding 0.999,999,999,999,999,999,999,999,99. In conclusion, the Microreader 23HS Plex ID System shows promise as a valuable supplementary tool for forensic applications, particularly in addressing complex kinship testing and challenges posed by STR mutation.

Genetic polymorphism and forensic efficiency of 21 autosomal STR loci from Shandong Han population in Northern China.

BACKGROUND: Highly polymorphic autosomal STR loci are useful for understanding population structure better and for forensic application, however the non-CODIS STR loci in the Han population of Shandong, located in Northern China, are not well-characterised. AIM: To investigate population genetic polymorphism and forensic efficiency of 21 autosomal STR loci from the Shandong Han population in Northern China and reveal the genetic relationships with other populations both at home and abroad. SUBJECTS AND METHODS: In this study, population genetic data of 21 autosomal STR loci included in the Goldeneye DNA ID 22NC Kit that includes four CODIS loci and 17 non-CODIS loci were determined for 523 unrelated Han individuals in Shandong. RESULTS: Significant deviations from Hardy-Weinberg equilibrium were not observed. A total of 233 alleles were detected with allele frequencies ranging from 0.0010 to 0.3728. The combined power of discrimination was 0.99999999999999999999999990011134, and the combined power of exclusion was 0.99999999788131. Furthermore, in an analysis of population differentiation Nei's standard genetic distance and multidimensional scaling analysis, which were conducted based on the overlapping 15 STR loci, revealed that the Shandong Han population was most closely related to populations in close geographic proximity. CONCLUSIONS: This study demonstrated that the 21 autosomal STR loci included in the GoldeneyeTM DNA ID 22NC system are highly polymorphic and suitable for forensic identification and paternity testing in the Shandong Han population. Additionally, the present results enrich the population genetic database.

Multiple genetic analyses for Chinese Hunan Han population via 46 A-STRs.

BACKGROUND: Short tandem repeats (STRs) are important as common genetic markers in forensic identification and population genetics due to their highly polymorphic nature. AIMS: To explore genetic polymorphisms of the Chinese Hunan Han population and further dissect genetic relationships among the Hunan Han and other populations from China. SUBJECTS AND METHODS: In this study, samples of 394 unrelated healthy individuals from the Chinese Hunan Han population were analysed using 46 autosomal-STRs (A-STRs). Thirteen previously reported populations (6378 individuals) from China were subsequently collected for population genetic analyses based on 23 shared A-STRs. RESULTS: In the Hunan Han population, a total of 452 alleles were detected in 46 A-STRs with allelic frequencies spanning from 0.0013 to 0.5571. Except for the Penta D locus in linkage disequilibrium, the combined power of discrimination and the combined power of exclusion for 45 A-STRs in the Hunan Han population were 0.999999999999999999999999999999999999999999999999510314 and 0.999999999999999726596, respectively. Results of interpopulation differentiation, principal component analysis, and phylogenetic relationship analyses uniformly showed that the Hunan Han have closer genetic affinities with Han populations from different Chinese regions and a geographically close ethnic minority group, namely the Hubei Tujia. CONCLUSION: To summarise, these 46 A-STRs showed high polymorphism in the Chinese Hunan Han population for forensic practice.

Useful autosomal STR marker sets for forensic and paternity applications in the Central Indian population.

BACKGROUND: Many countries have developed their core set of STR loci for forensic application and database generation, which India lacks. AIM: To assess the usefulness of various combinations of autosomal STR marker sets for their superior use in the central Indian population for forensic and paternity applications. SUBJECTS AND METHODS: 19 STR marker sets were analysed on 200 central Indian populations and 20 paternity cases to assess their usefulness. RESULTS: Two marker sets each comprising 19 STR markers are found to be superior to 20 expanded CODIS loci in the studied population. These marker sets also showed their effectiveness in 20 paternity cases having CPI values of 7.62 × 1011 and 7.16 × 1011. Three non-CODIS STR markers Penta E, Penta D, and SE33 showed amplification in 50 challenging samples with >0.80 heterozygosity. CONCLUSION: Population-specific STR marker sets are useful in forensic and paternity applications, as well as database generation, and it is envisioned that Penta E, Penta D, and SE33 markers will be included in the list of core STR loci in the central Indian population.

Population genetic data of 22 autosomal STR loci for the Scheduled Caste population of Rajasthan India.

AIM: To study the genetic structure of the Scheduled Caste population of Rajasthan and its relationship with Indian and global populations using expanded 20 CODIS STR loci (autosomal) markers. SUBJECTS AND METHODS: Blood samples of 226 healthy, unrelated adult individuals of the Scheduled Caste population of the Indian state of Rajasthan were taken from the routine casework of authors after obtaining written informed consent. Autosomal STR markers included in PowerPlex® Fusion 5 C and GlobalFiler™ PCR amplification kits were used to explore the genetic diversity of the studied population. Amplicons were separated using Genetic Analyser 3500XL as per the recommended protocol. RESULTS: Observed heterozygosity for the studied population ranged from 0.681(CSF1PO) to 0.881 (D1S1656).Combined Discrimination Power and Combined Exclusion Power were observed as 1 and 0.9999999852, respectively. The highest Discrimination Power was observed for the locus D1S1656. In the population comparison test, Nei's Da distance-based Neighbor-Joining (NJ) dendrogram revealed two significant clusters of geographically close Indian and East Asian populations along with a few small groups of outlier populations. CONCLUSION: The matching probability for 20 STR markers was observed as 7.02 × 10-24 and paternity index as 5.55 × 107. These values play a key role in forensic applications.The studied population showed a higher genetic affinity with geographically closer populations than the distant ones. This caste-based population data is expected to play an important role in forensic DNA applications and genetic studies.

Weight-of-evidence for DNA identification of missing persons and human remains using CODIS.

DNA Identification of unidentified human remains (UHR) is performed in Israel by comparing the UHR's short tandem repeat (STR) profiles to a national database of STR profiles taken from relatives of missing persons. Kinship analysis is performed using the CODIS 7.0 software and results are stated as a Joint Pedigree Likelihood Ratio (JPLR). The weight-of-evidence for JPLR has never been studied, making it difficult to interpret the meaning of specific values in terms of whether UHR are related to specific pedigrees. Therefore, the aim of this study was to statistically determine the practical meaning and context of the JPLR. We used 440 million pairs of simulated DNA profiles and 294 pairs of real ones from known siblings, parent/offspring and unrelated persons. A Score-Based Likelihood Ratio (SBLR) was empirically constructed, validated and compared to both JPLR and the LR produced by CODIS. Our results show that CODIS's JPLR and LR values for single-person pedigrees overestimate the level of support for both "parent/child" and "siblings" propositions relative to the "unrelated" proposition, by up to two orders of magnitude. A practical table is given for correcting this phenomenon, with statistical interpretation (i.e. SBLR) for each JPLR score, including verbal levels of propositional support ranging from "no support" (SBLR<2) to "extremely strong" (SBLR>1 Million).

Development of a new 25plex STRs typing system for forensic application.

We have developed a novel STR 25-plex florescence multiplex-STR kit (DNATyper25) to genotype 23 autosomal and two sex-linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non-CODIS. The sex-linked markers include a Y-STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non-human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/µL and hematin concentration <400 µM. For forensic evaluation, the 23 autosomal STRs followed the Hardy-Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10-22 , TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.

Allele frequencies and forensic genetic parameters of 10 supplementary and two CODIS loci in a Japanese population genotyped using an Investigator HDplex Kit.

A total of 344 unrelated Japanese adults were genotyped to determine allele frequencies and evaluate forensic parameters for 10 autosomal supplementary non-CODIS loci and 2 autosomal CODIS loci using an Investigator® HDplex Kit for complex relationship testing.

Genetic diversity of 23 STR loci in Guangxi Zhuang population and its phylogenetic relationship with 25 other populations.

Aim: To estimate genetic diversity of 23 STR loci included in the DNA TyperTM 25 Kit, and evaluate its effectiveness in forensic application.Subjects and methods: A total of 450 (251 males and 179 females) unrelated healthy individuals from Guangxi Zhuang population were amplified with DNA TyperTM 25 Kit, isolated by the 3730 Series Genetic AnalyzerTM, and genotyped using the GeneMapper ID-X. Genetic parameters and population relationships were analysed.Results: Allele frequencies ranged from 0.001 to 0.5889. The combined power of discrimination (CPD) and the combined power of exclusion (CPE) of the 23 STR loci were 0.999999999999999999 and 0.999996765, respectively. No deviations from Hardy-Weinberg equilibrium and linkage disequilibrium were observed. Inter-population comparison based on Fst, PCA, genetic distance, phylogenetic trees, and MDS showed that Zhuang population clustered with the populations holding a close geographic distance with Zhuang (Guangdong Han and Hainan Li populations).Conclusions: Our study indicated that the 23 autosomal STR loci included in DNA TyperTM 25 Kit can be used as forensic tools for individual identification and parentage testing. Moreover, the result of our mass investigation will enrich the forensic database of Chinese populations and serve for further study of the origin of anthropology.

Familial DNA searching- an emerging forensic investigative tool.

In recent years, jurisdictions across the United States have expressed a growing interest in aiding criminal investigations through the use of familial DNA searching (FDS)- a forensic technique to identify family members through DNA databases. The National Survey of CODIS Laboratories surveyed U.S. CODIS laboratories about their perceptions, policies, and practices related to FDS. In total, 103 crime labs completed the survey (77% response rate). Labs in 11 states reported using FDS, while labs in 24 states reported using a similar-but distinct- practice of partial matching. Although the majority of labs had positive perceptions about the ability of FDS to assist investigations, labs also reported a number of concerns and challenges with implementing FDS. Respondents reported using either practice a limited amount with modest numbers of convictions resulting from both FDS and partial matching. The article reports on varying practices related to official policies, training, eligibility, the software search, lineage testing, requirements for releasing information, and subsequent investigative work. Finally, the article discusses what can be learned from this survey, accompanying limitations, and implications for decision-makers considering using FDS.

Developmental validation of GlobalFiler™ PCR amplification kit: a 6-dye multiplex assay designed for amplification of casework samples.

The GlobalFiler™ PCR Amplification Kit is a single multiplex assay that amplifies a set of 24 markers, which encompass the European Standard Set and CODIS (Combined DNA Index System) recommended composite set of loci. In addition to more loci and a 6-dye chemistry format, the Master Mix has been formulated to allow higher sample loading volume for trace DNA samples. The GlobalFiler™ Kit has been optimized to deliver high performance on casework samples, while also delivering fast thermal cycling, with an amplification time of approximately 80 min. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit's robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

Population Identifiability from Forensic Genetic Markers: Ancestry Variation in Latin America.

The Combined DNA Index System (CODIS) loci comprise a standard microsatellite marker set widely used for distinguishing among individuals in forensic DNA identity testing for medicolegal casework in the United States and in other countries. In anthropological genetic research, CODIS markers have become an important tool for uses extending beyond case investigations to quantify ancestry proportions, reveals patterns of admixture, and trace population histories. These investigations are especially prevalent in studies of Latin American population structure. Nevertheless, the accuracy of the ancestry estimates computed from the CODIS loci for highly admixed Latino populations has not been formally tested. Longstanding arguments have been made that small ancestry panels, including the CODIS loci specifically, are not suitable for ancestry inference in admixed populations, due to high heterozygosity and limited number of loci used. Recent studies on ancestry inference using the CODIS loci suggest that these do confer more information of population-level identifiability than recognized in forensic genetic scholarship and by the medicolegal community. Here, we formally test the ability of CODIS and CODIS-proxy (e.g., high-heterozygosity and individual-identifiability loci) marker panels to accurately estimate admixture proportions of individuals, including a sample of Latinos with a wide range of ancestry proportions. Using the same individuals to make direct comparisons of the outcomes, the authors produced ancestry estimates from (a) a small CODIS/CODIS-proxy locus panel and (b) a robust and validated microsatellite ancestry-informative panel. They found evidence (e.g., ρ = 0.80-0.88) that supports the use of CODIS/ CODIS-proxy loci to capture the general ancestry estimation trends of a sample. This finding is in line with results of studies using CODIS on Latin American populations: the ancestry estimations generated by CODIS present trends supported by documented population histories (e.g., colonialism and population movements) and microevolutionary events (e.g., gene flow) in Latin America. However, this study also highlights the limitations of CODIS for making individual-level inferences of ancestry: the associated estimates for an acceptable level of statistical confidence (95%) are too broad to make any nuanced inferences regarding an individual's actual ancestry composition.

FlexPlex27-highly multiplexed rapid DNA identification for law enforcement, kinship, and military applications.

Rapid DNA identification is the use of a rugged, field-deployable system to generate short tandem repeat (STR) profiles in law enforcement, military, immigration, and homeland security applications. A performance verification study was conducted on the ANDE Rapid DNA identification system using FlexPlex27, a highly multiplexed, 27 locus assay that generates data for the expanded CODIS core loci and all additional STR loci required for international databasing. The assay contains 23 autosomal loci (D1S1656, D2S1338, D2S441, D3S1358, D5S81, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, CSF1PO, Penta E, TH01, vWA, TPOX, and SE33), three Y-chromosomal loci (DYS391, DYS576, and DYS570), and Amelogenin. Study results demonstrate that the instrument is reliable, reproducible, accurate, robust, and ready for a large scale, comprehensive developmental validation by NDIS-participating laboratories. The additional loci in the FlexPlex assay allow for improved STR profile sharing globally, increase the power of discrimination for identification matches, and improve the effectiveness of kinship analyses.

Population data of 13 nonCODIS STR markers located inside the 6 nonsyndromic oculocutaneous albinism genes.

Allele frequencies and forensic statistics of 13 autosomal short tandem repeat loci were estimated in 56 unrelated Iranian individuals. Except for two loci which were monomorph, a total of 5-11 alleles at each locus were observed and altogether 94 alleles for all selected loci were found. Our results show that 11 out of 13 nonCODIS STR loci are polymorphic and can be useful for human identification and kinship analysis and relationship investigations in Iran.

Comprehensive landscape of non-CODIS STRs in global populations provides new insights into challenging DNA profiles.

The worldwide implementation of short tandem repeats (STR) profiles in forensic genetics necessitated establishing and expanding the CODIS core loci set to facilitated efficient data management and exchange. Currently, the mainstay CODIS STRs are adopted in most general-purpose forensic kits. However, relying solely on these loci failed to yield satisfactory results for challenging tasks, such as bio-geographical ancestry inference, complex DNA mixture profile interpretation, and distant kinship analysis. In this context, non-CODIS STRs are potent supplements to enhance the systematic discriminating power, particularly when combined with the high-throughput next-generation sequencing (NGS) technique. Nevertheless, comprehensive evaluation on non-CODIS STRs in diverse populations was scarce, hindering their further application in routine caseworks. To address this gap, we investigated genetic variations of 178 historically available non-CODIS STRs from ethnolinguistically different worldwide populations and studied their characteristics and forensic potentials via high-coverage whole genome sequencing (WGS) data. Initially, we delineated the genomic properties of these non-CODIS markers through sequence searching, repeat structure scanning, and manual inspection. Subsequent population genetics analysis suggested that these non-CODIS STRs had comparable polymorphism levels and forensic utility to CODIS STRs. Furthermore, we constructed a theoretical next-generation sequencing (NGS) panel comprising 108 STRs (20 CODIS STRs and 88 non-CODIS STRs), and evaluated its performance in inferring bio-geographical ancestry origins, deconvoluting complex DNA mixtures, and differentiating distant kinships using real and simulated datasets. Our findings demonstrated that incorporating supplementary non-CODIS STRs enabled the extrapolation of multidimensional information from a single STR profile, thereby facilitating the analysis of challenging forensic tasks. In conclusion, this study presents an extensive genomic landscape of forensic non-CODIS STRs among global populations, and emphasized the imperative inclusion of additional polymorphic non-CODIS STRs in future NGS-based forensic systems.

Lead with speed: Recasting the forensic laboratory product line and service delivery model.

Forensic laboratories face a multitude of challenges when striving to deliver services to the criminal justice system. While many of these issues change over time, one in particular seems to endure the test of time … the need for faster results. Law enforcement wants and needs quicker response times to access critical information required to investigate their cases. One answer to this persistent problem is evolving technology. Technology not only permits a much quicker response than forensic laboratories are currently delivering, it can open the door to solving previously unsolvable cases. Along with applying new technology, an evaluation of current forensic laboratory product lines, service delivery models, and mindset regarding the role of forensic science-based investigative leads (termed forensic leads) is warranted. Resources and strategic planning are needed to realize the full potential of evolving technologies and what forensic laboratories can do to provide actionable and timely forensic leads to our criminal justice partners as a normal course of action instead of as an exception. This proposal is to establish a permanent, designated Forensic Lead Program (FLP) that resides under the umbrella of an accredited forensic laboratory and is tasked with the development and release of forensic leads. The FLP involves a focused menu of services, defined personnel roles, strict protocols, short turnaround time, standardized expectations, and targeted training, combined with the sense of urgency needed for consistent delivery of timely and actionable forensic leads. A dedicated FLP will save time and money by providing critical information for more focused investigations. 'Speed is the need' … for quick identification of those that threaten public safety and for the equally quick elimination of those wrongfully accused. Programs at two large state forensic laboratories will demonstrate how these concepts could be implemented along with their learning experiences. A business case will also be included to demonstrate the cost benefit of the Forensic Lead Program for DNA (CODIS - Combined DNA Index System) and NIBIN (National Integrated Ballistic Information Network), however other section services are expected to see similar benefits. Improving the response time by one day saves $1677.75 per $1 spent [1]. The return on investment (ROI) for applying DNA to firearms evidence returns $47.88 per $1 spent, or an 4,788 % ROI. Applying NIBIN (National Integrated Ballistic Information Network) to firearms evidence to provide investigative leads is $502.19 per $1 spent, which is a 50,219 % ROI. Recasting the forensic laboratory product line and service delivery model to 'Lead with Speed' makes both economic and investigative sense.

Eligible offender samples are missing in CODIS: A statewide approach to performing a lawfully owed DNA census.

In 2019, the Texas Department of Public Safety (TXDPS) Texas Ranger Division (TRD) identified approximately 3300 registered sex offenders (RSOs) from whom a "lawfully owed" DNA sample was missing from the Federal Bureau of Investigation's Combined DNA Index System (CODIS). Lawfully owed DNA (LODNA) is defined as a DNA sample from a qualifying offender who should have had their sample entered into CODIS, but for unknown reasons did not. As a result of those findings, TXDPS then applied for and was awarded a grant from the Bureau of Justice Assistance's Sexual Assault Kit Initiative to collect DNA specimens from these RSOs, and to perform a statewide LODNA census. TXDPS TRD sought to determine: Are the missed DNA collection problems limited to RSO's or are they occurring among individuals with a qualifying arrest or conviction as specified by state law too? What processes are used to identify individuals who are eligible for DNA sample collection? How is an individuals' DNA collection eligibility conveyed to external agencies? The findings from TXDPS' LODNA census, identified 43,245 individuals who were likely eligible for DNA collection between 1995 and 2020, therefore indicating statewide DNA collection issues. Over 4 years, collection efforts pertaining to the aforementioned lawfully owed census, have yielded 5183 LODNA sample collections, and 276 CODIS hits. This manuscript aims to create an awareness within other agencies of the importance of implementing best practices to ensure the collection and upload of LODNA from every eligible individual.

A new search tool for familial searches in the Israel Criminal DNA database.

The Israel DNA database has recently started to conduct familial searches (FS). We adopted the CODIS pedigree strategy for FS, which is used in the Unidentified Human Remains (UHR) database and implemented it into the criminal forensic database. This strategy is based on Kinship analysis performed in pedigrees containing DNA profiles from the crime scene designated "unknown," that are then searched against the entire suspects database. A list of candidates is produced and ranked by Joint Pedigree Likelihood Ratio (JPLR). Traditional Y-STR characterizing and mitochondrial sequencing can be performed in order to further minimize the list. Our novel strategy consists of an additional pedigree analysis aimed at prioritizing potential candidates from the candidate list: a Test Pedigree Tree (TPT). Candidates ranked high on the JPLR list can be verified or eliminated from the list by using other close family members included in the database. To further validate this novel strategy, we describe two cases where implementation of this strategy led to a successful match and solved the crime.

Efficiency evaluation of common forensic genetic markers for parentage identification involving close relatives.

To explore the efficacy of commonly used forensic identification panels in complex paternity testing of trios that involved close relatives, we wrote a code by R to generate 10,000 pedigrees at 20 CODIS STR, 21 non-CODIS STR and 30 InDel loci in Chinese five ethnic groups based on their allele frequencies. Parentage identification index--cumulative paternity index (CPI) value was set as output and was further analyzed to evaluate the performance of the aforementioned panels in complex paternity testing when the alleged parent is a random individual, biological parent, grandparent, sibling of biological parent, half-sibling of biological parent, etc. The results showed that the false inclusion of parent sibling posed as parent demonstrated no statistically significant difference from that of grandparent posed as parent. The scenarios where both biological parent and alleged parent were consanguineous to the other parent were also simulated. The results revealed that the complexity of paternity testing would raise when biological parents were consanguineous and the alleged parent was a close relative of theirs. Despite the values of non-conformity number could vary in different genetic relationships, populations and panels, 20 CODIS STRs and 21 non-CODIS STRs performed satisfactorily in most simulated scenarios. However, the joint use of 20 CODIS STRs and 21 non-CODIS STRs is more recommendable when resolving the paternity testing of the incest mating case. Overall, the current study could be regarded as a worthwhile reference in complex paternity testing of trios that involved close relatives.