Development and validation of a novel SiFaSTRTM 23‐plex system

Human identification and paternity testing are usually based on the study of STRs depending on their particular characteristics in the forensic investigation. In this paper, we developed a sensitive genotyping system, SiFaSTR^? 23‐plex, which is able to characterize 18 expanded Combined DNA Index System STRs (D3S1358, D5S818, D2S1338, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D10S1248, D8S1179, D1S1656, D18S51, D12S391, D19S433, D16S539, D13S317, and FGA), three highly polymorphic STRs among Chinese people (Penta D, Penta E, and D6S1043), one Y‐chromosome Indel and amelogenin using a multiplex PCR; the PCR amplified products were analyzed using the Applied Biosystems 3500 Genetic Analyzer. Full genotyping profiles were obtained using only 31.25 pg of control DNA; various case‐type specimens, as well as ten‐year‐old samples were also successfully genotyped. Additionally, in the validation studies, this multiplex was demonstrated to be human‐specific and compatible with the interference of multiple PCR inhibitors. The system also enabled the detection of mixtures, and complete profiles could be obtained at the mixed ratios of 1:1, 1:3, and 3:1. The development and validation study here illustrated that the SiFaSTR^? 23‐plex system is accurate, powerful, and more sensitive than many other systems. What's more, the population data in our study not only illustrated that this 23‐plex typing system was straightforward and efficient but also expanded the Chinese STR database, which could facilitate the appropriate application of the 23 genetic markers in forensic genetics, especially in the Chinese population.     

Massively parallel sequencing of STRs using a 29-plex panel reveals stutter sequence characteristics

Massively parallel sequencing of forensic STRs simultaneously provides length-based genotypes and core repeat sequences as well as flanking sequence variations. Here, we report primer sequences and concentrations of a next-generation sequencing (NGS)-based in-house panel covering 28 autosomal STR loci (CSF1PO, D1GATA113, D1S1627, D1S1656, D1S1677, D2S441, D2S1776, D3S3053, D5S818, D6S474, D6S1017, D6S1043, D8S1179, D9S2157, D10S1435, D11S4463, D13S317, D14S1434, D16S539, D18S51, D18S853, D20S482, D20S1082, D22S1045, FGA, TH01, TPOX, and vWA) and the sex determinant locus Amelogenin. Preliminary evaluation experiments showed that the panel yielded intralocus- and interlocus-balanced sequencing data with a sensitivity as low as 62.5 pg input DNA. A total of 203 individuals from Yunnan Bai population were sequenced with this panel. Comparative forensic genetic analyses showed that sequence-based matching probability of this 29-plex panel reached 2.37 × 10⁻²⁹, which was 23 times lower than the length-based data. Compound stutter sequences of eight STRs were compared with parental alleles. For seven loci, repeat motif insertions or deletions occurred in the longest uninterrupted repeat sequences (LUS). However, LUS and non-LUS stutters co-existed in the locus D6S474 with different sequencing depth ratios. These results supplemented our current knowledge of forensic STR stutters, and provided a sound basis for DNA mixture deconvolution.     

DNA sequencing and multiplex STR analysis on plastic microfluidic devices

The ability of plastic microfluidic devices with separation channel lengths of 6, 10 or 18 cm to perform high-quality and high-performance ssDNA analysis was evaluated. Specifically, four-color DNA sequencing separation of a terminator sequencing standard using replaceable, urea-denaturing linear polyacrylamide (LPA) solution as a sieving matrix, yielded read lengths of 410 bases in 15 min with base calling accuracy of 99.2% on a 6-cm device, and 640 bases in 35 min with accuracy of 98.0% on a 18-cm device. A two-color sizing analysis of four-locus (CSF1PO, TPOX, TH01, vWA) short tandem repeats (STRs) allelic ladder on a 10-cm device indicated a mean SD of +/- 0.08 base pairs (bp) between runs, and single bp resolution of spiked TH01 allele 9.3 (198 bp) from TH01 allele 10 (199 bp) of the CTTv ladder with R = 0.81. A four-color multiplex sizing analysis of three different AmpFlSTR allelic ladders consisting of nine loci (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) and gender alleles (Amelogenin) on a 10-cm device had a mean SD of +/- 0.15 bp between runs for sizing three loci, i.e., FGA, D18S51 and D3S818; alleles differing by 2 bp in size were resolved with resolutions close to baseline. This work demonstrates that plastic microfluidic devices are capable of quality sequencing and STR sizing comparable to that of glass devices of similar separation lengths.     

Slippage mutation rates in 15 autosomal short tandem repeat loci for forensic purposes in a Southeastern Brazilian population

Well‐defined estimates of mutation rates in highly polymorphic tetranucleotide STR loci are a prerequisite for human identification in genetics laboratory routines useful for civil and criminal investigations. Studying 15 autosomal STR loci of forensic interest (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, and vWA), we detected 193 slippage mutations (189 one‐step and four two‐step mutations) in 148 875 parent‐child allelic transfers from 5171 paternity cases with true biological relationship (15 096 individuals; 4754 trios and 417 duos; 9925 meiosis) from the state of São Paulo, a very representative population of Brazil. The overall mutation rate was 1.3 × 10^?3 and the highest rates were observed at loci vWA (2.8 × 10^?3), FGA and D18S51 (2.7 × 10^?3 for both), while loci TH01 and TPOX did not present any mutations. The mean slippage mutation rate of paternal origin (1.8 × 10^?3) was six times higher than that observed for maternal origin (0.3 × 10^?3).     

Genetic polymorphisms of 20 short tandem repeat loci from the Han population in Henan,China

The aim of this study was to investigate the genetic polymorphism of 20 short tandem repeat (STR) loci including D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA in Han population of Henan, China and to assess its value in forensic science. Genomic DNA was extracted from 274 blood samples of unrelated healthy individuals in the Henan Han population. Alleles were amplified with PowerPlex® 21 system kit and PCR products were detected with ABI3130 genetic analyzer (Applied Biosystems) and the data were analyzed with modified PowerStats v1.2. A total of 229 alleles were observed in this Han population and the allelic frequencies ranged from 0.0020 to 0.5090 in the present study. Observed genotype distributions for each locus do not show deviations from Hardy–Weinberg equilibrium expectations (p < 0.05). The combined power of discrimination, combined power of exclusion, and combined matching probability of this 20 STR loci were 0.999999999, 0.999999994603, and 4.0433 × 10^?24, respectively. The 20 STR loci are highly polymorphic in the Han population of Henan, China and they may be of great value in forensic science and human population genetics.     

Infrared fluorescent automated detection of thirteen short tandem repeat polymorphisms and one gender-determining system of the CODIS core system

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDye800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.     

Developmental validation of the Microreader™ 20A ID system

The Microreader? 20A ID system is designed for forensic applications such as personal identification, parentage testing, and research. It includes 13 combined DNA index system (CODIS) short tandem repeat (STR) loci (CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11), three expanded CODIS STR loci (D12S391, D19S433, and D2S1338), three non‐CODIS STR loci (D6S1043, Penta D, and Penta E), and the amelogenin locus in one reaction with a six‐dye fluorescent (FAM, HEX, TAMAR, ROX, PUR, and QD550) analysis system. In this study, the Microreader? 20A ID system was validated according to the Scientific Working Group on DNA Analysis Methods validation guidelines for forensic DNA Analysis methods and Chinese national standard, including PCR‐based studies, sensitivity study, precision, and accuracy evaluation, stutter calculation, inhibitor tests, species specificity, and DNA mixture studies. Our results suggest that the Microreader? 20A ID system is a useful tool for personal identification and parentage testing.     

Development and validation of a forensic six-dye multiplex assay with 29 STR loci

This paper describes the development and validation of a novel 31-locus, six-dye STR multiplex system, which is designed to meet the needs of the rapidly growing Chinese forensic database. This new assay combines 20 extended-CODIS core loci (D3S1358, D5S818, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D8S1179, D18S51, D16S539, D13S317, FGA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045), nine highly polymorphic loci in Chinese Han population (D3S3045, D6S1043, D6S477, D8S1132, D10S1435, D15S659, D19S253, Penta D, and Penta E), and two gender determining markers, amelogenin and Y-Indel, which could amplify DNA from extracts, as well as direct amplification from substrates. To demonstrate the suitability for forensic applications, this system was validated by precision and accuracy evaluation, concordance tests, case sample tests, sensitivity, species specificity, stability, stutter calculation, and DNA mixtures, according to the guidelines described by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and regulations published by the China Ministry of Public Security. The validation results indicate the robustness and reliability of this new system, and it could be a potentially helpful tool for human identification and paternity testing in the Chinese population, as well as facilitating global forensic DNA data sharing.     

X chromosomal recombination—A family study analyzing 26 X‐STR Loci in Chinese Han three‐generation pedigrees

The aim of this study is to investigate genetic linkage and recombination fractions of 26 X chromosomal (X‐STR) loci with two multiplex PCR systems (MX15‐STR and MX12‐STR). MX15‐STR (including DXS7133, DXS6801, DXS981, DXS6809, DXS7424, DXS6789, DXS9898, DXS7132, GATA165B12, DXS101, DXS10075, DXS6800, GATA31E08, DXS10074, and DXS10079) and MX12‐STR (including DXS6854, DXS9902, DXS6800, GATA172D05, DXS7423, HPRTB, DXS6807, DXS6803, DXS6804, DXS6799, DXS8378, and DXS8377) were successful analyzed on 206 two‐generation families with two or more children and 33 three‐generation families with 72 grandsons. Segregation analysis and calculation of recombination fractions between pairs of markers were performed. Linkage analysis of pairs of markers showed that there existed significant linkage (maximum LOD scores >2.0) within the physical distance of 48.5 Mb. Recombination events could be observed within the clusters of closed linked makers spanning <1.0 Mb. These results indicate that close cluster X‐STRs used and recombination fractions of the selected loci will be very useful for biostatistical calculations in complex kinship analysis.     

Validation of the Microreader 28A ID System: A 6-dye multiplex amplification assay for forensic application

The Microreader 28A ID System is a new 28-plex genotyping system with 6-dye multiplex amplification, which allows the simultaneous amplification of all 20 Combined DNA Index System (CODIS) core loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, D22S1045), plus five extended STRs loci (D6S1043, Penta D, Penta E, DYS391, SE33), 2 Y-Indels (Rs2032678, Rs771783753), and the amelogenin loci. This system can be used for forensic analyses, such as personal identification, kinship testing, scientific research, database applications, and other aspects of human genetic identification. The validation of the Microreader 28A ID System followed the “Validation Guidelines for DNA Analysis Methods (2016)” described by the Scientific Working Group on DNA Analysis Methods and the regulations published by the China Ministry of Public Security. Our tests included PCR-based studies, sensitivity study, precision and accuracy evaluation, stutter percentage and heterozygous peak height ratio, inhibitor tests, species specificity, and population studies. The validation results suggest that the Microreader 28A ID system is a robust and reliable amplification kit for personal identification, kinship testing, and forensic database applications.     

Application of electrophoresis technology to DNA analysis

We used the variable number tandem repeat (VNTR) polymorphism and the ten short tandem repeat (STR) polymorphisms to study a number of disputed paternity cases in the Japanese population. For the determination of VNTR locus (D1S80) and the ten STR loci (vWA, F13B, TH01, TPOX, CSF1PO, F13A01, LPL, D3S1744, D12S1090, D18S849) we used polymerase chain reaction (PCR) amplification and the vertical polyacrylamide gel electrophoresis technique followed by SYBR green I staining. The irregular repeats were analyzed by sequencing from bands of vertical polyacrylamide gel electrophoresis using the latest gene analyzing equipment, the ABI PRISM 310 Genetic Analyzer. The probable genotypes of the deceased putative father were deduced by Komatu's method from the genotypes of the widow and the genotypes of their children. The calculation of paternity probability used the Essen-Moller formula and Bayes's theorem. Calculated in eleven loci, the distinguishing probabilities (DP) and the mean exclusion chance (MEC) were 0.9999 and 0.9989, respectively. Therefore, information obtained from eleven DNA polymorphisms is enough to determine paternity plausibility.     

Standardized genotyping of HLA STR by CE as surrogate for HLA class I and II markers and for identification of HLA identical siblings

Linkage disequilibria (LD) between alleles and haplotypes of human leucocyte antigen, locus A (HLA) and STR loci located in the human major histocompatibility complex were analyzed in order to investigate whether or not HLA alleles and haplotypes are predictable by alleles or haplotypes of HLA STRs. Standardized genotyping of eight STR loci (D6S2972, D6S2906, D6S2691, D6S2678, D6S2792, D6S2789, D6S273, and DQIV) was performed by CE on 600 individuals from 150 Austrian Caucasoid families with known HLA‐A,‐B,‐C and –DRB1 typing. From those, 576 full haplotypes of four HLA and eight STR loci were obtained. Haplotypes of two flanking STRs predicted HLA alleles and two‐locus HLA haplotypes better than single STR alleles, except HLA‐DRB1 alleles (92% were in LD with DQIV alleles only). A percentage of 65–86% of three and four‐locus HLA haplotypes were in LD with haplotypes of three, four, and eight of their flanking STR loci including numerous clear‐cut predictions (20–61%). All eight and a set of the four most informative STR loci D6S2972, D6S2678, D6S2792, and DQIV could identify all HLA identical and nonidentical siblings in 138 pairs of siblings. The results of this proof of concept study in Austrian Caucasoids show, that HLA STRs can aid the definition of HLA‐A,‐B,‐C,‐DRB1 haplotypes and the selection of sibling donors for stem cell transplantation.     

Diversity study of 12 X‐chromosomal STR loci in Hui ethnic from China

X‐chromosomal STRs (X‐STRs) have been used as complements of autosomal STR application in recent years. In this work, we present population genetic data of 12 X‐STRs including DXS101, DXS10159, DXS10162, DXS10164, DXS6789, DXS7133, DXS7423, DXS7424, DXS8378, DXS981, GATA165B12, and GATA31E08 loci in a sample of 231 unrelated healthy individuals from the Hui ethnic group in Ningxia Hui Autonomous Region, China. Allelic frequencies of the 12 X‐STR loci and haplotypic frequencies of the reported linkage groups (DXS7424‐DXS101 and DXS10159‐DXS10164‐DXS10162) were investigated in the group, respectively. No STR loci showed significant deviations from the Hardy–Weinberg equilibriums and no linkage disequilibriums of pairwise loci were found after Bonferroni correction, respectively. A combined power of discrimination in female individuals was 0.999999999985 and that in male individuals was 0.99999967, respectively. The combined mean exclusion chance in deficiency cases, normal trios and duo cases were 0.999934, 0.995754, and 0.999796, respectively. Significant differences were observed from 0 to 8 loci, when making comparisons between the data of Hui ethnic group and previously reported data from other 16 populations. The results indicated the new panel of 12 X‐STR loci might be useful for forensic science application.     

Evaluation of the protective capabilities of nucleosome STRs obtained by large‐scale sequencing

Partial DNA profiles are often obtained from degraded forensic samples and are hard to analyze and interpret. With in‐depth studies on degraded DNA, an increasing number of forensic scientists have focused on the intrinsic structural properties of DNA. In theory, nucleosomes offer protection to the bound DNA by limiting access to enzymes. In our study, we performed large‐scale DNA sequencing on nucleosome core DNA of human leucocytes. Five nucleosome short tandem repeats (STRs) were selected (including three forensic common STRs (i.e. TPOX, TH01, and D10S1248) and two unpublished STRs (i.e. AC012568.7 and AC007160.3)). We performed a population genetic investigation and forensic genetic statistical analysis of these two unpublished loci on 108 healthy unrelated individuals of the HeBei Han population in China. We estimated the protective capabilities of five selected nucleosome loci and MiniFiler? loci with artificial degraded DNA and case samples. We also analyzed differences between sequencing results and software predicted results. Our findings showed that nucleosome STRs were more likely to be detected than MiniFiler? loci. They were well protected from degradation by nucleosomes and could be candidates for further nucleosome multiplex construction, which would increase the chances of obtaining a better balanced profile with fewer allelic drop‐outs.     

Systematic assessment of the performance of Illumina's MiSeq FGx™ forensic genomics system

This study assesses the performance of Illumina's MiSeq FGx System for forensic genomics by systematically analyzing single source samples, evaluating concordance, sensitivity and repeatability, as well as describing the quality of the reported outcomes. DNA from 16 individuals (9 males/7 females) in nine separate runs showed consistent STR profiles at DNA input ≥400 pg, and two full profiles were obtained with 50 pg DNA input. However, this study revealed that the outcome of a single sample does not merely depend on its DNA input but is also influenced by the total amount of DNA loaded onto the flow cell from all samples. Stutter and sequence or amplification errors can make the identification of true alleles difficult, particularly for heterozygous loci that show allele imbalance. Sequencing of 16 individuals’ STRs revealed genetic variations at 14 loci at frequencies suggesting improvement of mixture deconvolution. The STR loci D1S1656 and DXS10103 were most susceptible to drop outs, and D22S1045 and DYS385a‐b showed heterozygote imbalance.  Most stutters were typed at TH01 and DYS385a‐b, while amplification or sequencing errors were observed mostly at D7S820 and D19S433. Overall, Illumina's MiSeq FGx System produced reliable and repeatable results.  aSTRs showed fewer drop outs than the Y‐ and X‐STRs.     

Structural polymorphism analysis of Chinese Mongolian ethnic group revealed by a new STR panel: Genetic relationship to other groups

Mongolian is the eighth largest ethnic minority on Chinese population data according to the 2010 census. In the present study, we presented the first report about the allelic frequencies and forensic statistical parameters at the 21 new STRs and analyzed linkage disequilibrium of pairwise loci in the Mongolian ethnic minority, China. Hardy–Weinberg equilibrium tests demonstrated no significant deviations except for the D1S1627 locus. The cumulative power of discrimination and power of exclusion of all the loci are 0.9999999999999999992576 and 0.9999997528, respectively. The results of analysis of molecular variance showed that significant differences between the Mongolian and the other eight populations were found at 1‐9 STR loci. In population genetics, the results of principal component analysis, structure analysis, and phylogenetic reconstruction analysis indicated shorter genetic distance between the Mongolian group and the Ningxia Han. All the results suggest that the 21 new STR loci will contribute to Chinese population genetics and forensic caseworks in the Mongolian group.     

Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing

There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population‐divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12‐STR multiplex composed of ancestry informative marker STRs (AIM‐STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM‐SNPs: Snipper, to handle multiallele STR data using frequency‐based training sets. We assessed the ability of the 12‐plex AIM‐STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM‐SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available.     

A powerful,novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations

A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)-Japanese and 187 Okinawa-Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy-Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu-Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu-Japanese were almost the same as in the Okinawa-Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.     

"Paterniplex", a highly discriminative decaplex STR multiplex tailored for investigating special problems in paternity testing

The goal of the study was to develop a STR multiplex ("Paterniplex") that is--as supplement to commercially available multiplex kits like the Identifiler kit (Applied Biosystems, Foster City, CA)--suitable for solving complex paternity cases such as deficiency cases or cases with mutations. The Paterniplex comprises the nine highly polymorphic STRs D8S1132, D7S1517, D10S2325, D12S391, Se33, D17S976, Penta E, Penta D and FGA in addition to Amelogenin as sex determination marker. The loci were selected because of their high degree of polymorphism (higher than that of the widely used TH01 marker). Only one locus, FGA, is shared with the Identifiler kit to avoid sample mix up. The study further gives details on the population genetics of the loci in a German Caucasian population (allelic distribution, Hardy-Weinberg Equilibrium and forensic efficiency markers such as the Discriminating Power) and three examples for cases that could not be solved using commercially available kits alone, but using the Paterniplex in addition to a commercial kit.     

Haplotype studies support slippage as the mechanism of germline mutations in short tandem repeats

Germline mutations of human short tandem repeat (STR) loci are expansions or contractions of repeat arrays which are not well understood in terms of the mechanism(s) underlying such mutations. Although polymerase slippage is generally accepted as a mechanism capable to explain most features of such mutations, it is still possible that unequal crossing over plays some role in those events, as most studies in humans could not exclude unequal crossing over (UCO). Crossing over can be studied by analyzing haplotypes using flanking markers. To check for UCO in mutations, we have analyzed 150 paternity cases for which more than the usual trio (mother, child, and father) were available for testing by analyzing 16 STR loci. In a total of 4900 parent-child allele transfers four mutations were observed at different loci (D8S1179, D18S51, D21S11, and SE33/ACTBP2). To identify the mutated allele and to check for UCO, we typed at least four informative loci flanking the mutated locus and used the pedigree data to establish haplotypes. By doing so we were able to exclude UCO in each case. Moreover, we were able to identify the mutations as one-repeat contractions/expansions. Our data thus support slippage as the mechanism of germline mutations in STRs.