Analysis of homozygous allele mismatches in paternity tests with massively parallel sequencing.

In paternity testing, short tandem repeats (STRs) allele mismatches are often detected. Nowadays, polymerase chain reaction- and capillary electrophoresis (CE)-based STR genotyping is the most commonly used method to distinguish alleles based on their length. However, it could not detect alleles of the same size with sequence differences. Massively parallel sequencing (MPS) can determine not only allele sizes but also sequences, which could explain the causes of allele mismatches. Additionally, more types of genetic markers can be detected in a single assay, which increases the discriminatory power and facilitates the analysis of paternity tests. In this study, we analyzed 11 cases with homozygous allele mismatches from routine DNA trio paternity tests using the CE platform. Samples were sequenced using the ForenSeq DNA Signature Prep Kit and the MiSeq FGx Sequencing System. The results show that of the eight father-child mismatch cases and three mother-child mismatch cases, five cases with D5S818 and D8S1179 and one case at D13S317 were classified as non-amplification. The other three cases and two cases could be defined as mutations. This study suggests that MPS-based STR genotyping can provide additional information that allows more accurate interpretation of allelic mismatches in paternity testing.

Genetic polymorphism analysis and forensic application evaluation of 57 insertion/deletion polymorphisms from Yi ethnic group in Yunnan.

BACKGROUND: As a new kind of diallelic genetic marker, insertion/deletion (InDel) polymorphisms have recently been used in forensic science. However, there are relatively few studies on the forensic evaluation of InDel genetic polymorphisms from different populations. AIM: The aim of the present work is to assess the genetic polymorphism and forensic applicability of 57 InDels from the Yi ethnic group and explore the genetic background of this group. SUBJECTS AND METHODS: A total sample of 122 unrelated individuals of Yi group from the Yunnan province were genotyped by the AGCU indel 60 Kit. Multiplex population genetic analyses on the same 57 InDels were carried out among the Yunnan Yi group and 29 reference populations. RESULTS: The average allele frequency of these loci in the Yi ethnic group was 0.485. Heterozygosity, polymorphism information content, and the power of discrimination were 0.477, 0.362, and 0.612, respectively. The combined power of discrimination and the combined power of exclusion reached to 0.99999999999999999669 and 0.999962965, respectively. The results showed that 57 InDels polymorphisms have high genetic polymorphisms in the Yi ethnic group. CONCLUSIONS: The 57 InDels could be used for forensic individual identification, paternity testing, and intercontinental population discrimination, with the potential for use in biogeographic ancestry inference.

A rare case of type II tri-allelic inheritance at vWA, SE33, D8S1179, and D13S317 loci demonstrated by STR analysis in paternity testing.

Short tandem repeat (STR) typing has been regularly used in paternity disputes and forensic human identification linked caseworks. Occasionally, forensic scientists come across aberrant allele patterns during STR typing because of mutations, genetic variations, and other abnormalities. The tri-allelic pattern of STR is rare, particularly, the case where this pattern exists at 4 loci. Here, we report the type II tri-allelic patterns observed at vWA, SE33, D8S1179, and D13S317 loci in the product of conception (POC) sample during the course of our regular paternity case investigation. The DNA extracted from the blood samples and tissue of POC were subjected to STR typing for autosomal and sex STR loci using the commercial QIAGEN's Investigator® IDplex Plus Kit and QIAGEN's Investigator® 24plex QS Kit. Capillary electrophoresis was carried out in 3500 and 3500xL Genetic Analyzer Applied Biosystems and genotyped using GeneMapper ID-X Software v1.5 and v1.6. In this case of paternity inclusion, the POC sample displayed type II tri-allelic patterns at vWA (16, 19, 20), SE33 (19, 28.2, 29.2), D13S317 (16, 19, 20), and D8S1179 (10, 13, 17) loci. In addition, the POC displayed an abnormal genotype with a heterozygous peak imbalance (type II-B) of (1:2) pattern at D3S1358, D21S11, and D16S539 loci, of (2:1) pattern at D1S1656, D12S391, D10S1248, D2S1338, D2S441, D18S317, FGA, CSF1PO, and D5S818 loci, and type II-C allelic pattern (one single peak with triplicate height) at D19S433 and DS7820 loci. Understanding of such anomalous genotypes improves the knowledge about tri-allelic pattern of CODIS loci and helps in the appropriate interpretation of the results in STR typing.

The Latest Research Progress on Cell-Free DNA and Prospects of Its Forensic Application.

In recent years, with the continuous progress of DNA extraction and detection technology, cell-free DNA(cfDNA)has been widely used in the life science field, and its potential application value in forensic identification is becoming more and more obvious. This paper reviews the concept, formation mechanism, and classification of cfDNA, etc., and describes the latest research progress of cfDNA in personal identification of crime scene touch DNA samples and non-invasive prenatal paternity testing (NIPPT). Meanwhile, this paper summarizes the potential application of cfDNA in injury inference, and discusses the advantages and disadvantages of common cfDNA analysis methods and techniques, and its application prospects, to provide a new idea for the wide application of cfDNA in the field of forensic science.

An Accidental Genetic Epidemiologist.

I briefly describe my early life and how, through a series of serendipitous events, I became a genetic epidemiologist. I discuss how the Elston-Stewart algorithm was discovered and its contribution to segregation, linkage, and association analysis. New linkage findings and paternity testing resulted from having a genotyping lab. The different meanings of interaction-statistical and biological-are clarified. The computer package S.A.G.E. (Statistical Analysis for Genetic Epidemiology), based on extensive method development over two decades, was conceived in 1986, flourished for 20 years, and is now freely available for use and further development. Finally, I describe methods to estimate and test hypotheses about familial correlations, and point out that the liability model often used to estimate disease heritability estimates the heritability of that liability, rather than of the disease itself, and so can be highly dependent on the assumed distribution of that liability.

Maternal uniparental disomy of chromosome 21 as a cause of pseudo-exclusion from paternity.

Uniparental disomy (UPD) is a rare chromosomal condition, which apart from its importance in medical genetics can affect an outcome of parentage DNA testing, often causing pseudo exclusions. We describe a case of trio paternity test using 24 informative STR loci with potential exclusion at 2 systems located on chromosome 21. Consequent genotyping of an additional 25 autosomal and 27 Y-specific STRs revealed one other inconsistency, also located on this chromosome. All three inconsistent markers had the same heteroallelic state between the child and the biological mother providing evidence for maternal heterodisomy of chromosome 21. The case highlights the importance of considering UPD as a cause of genetic inconsistencies, especially when the inconsistent marker systems are located on the same chromosome.

Calculation of the Paternity Index for the Alleged Father Related to the Child's Mother.

OBJECTIVES: To derive the paternity index (PI) calculation formula of the alleged father (AF) when the AF is a relative (parent/child, siblings, grandparent/grandchild, uncle/nephew, first cousins) of the child's biological mother. METHODS: For the case when the AF is related to the child's biological mother, the existence of the relationship in the numerator and denominator hypothesis of PI was considered. The genotype frequency of the AF was calculated by using the frequency formula in which the mother's genotype was considered, while the random male in the denominator was substituted as another relative of the mother's same rank. The PI calculation formula was derived to eliminate the effect of the relationship between AF and the child's biological mother. RESULTS: When the AF and the biological mother have first, second and tertiary kinship, a more conservative PI was obtained from the PI calculation formula derived in this study compared with the PI calculation method which did not consider kinship. CONCLUSIONS: The calculation method provided in this study can eliminate the effect of the relation of the AF and mother on the PI in incest cases, to obtain more accurate and conservative identification conclusions.

Mutational analysis of 16 STR markers in the Slovak population.

BACKGROUND: Short tandem repeats (STRs) are genetic markers frequently used for human identification and paternity testing. They are highly mutable, which may occasionally lead to inconsistencies between the genotypes of parents and their children. As the mutation rates of individual STR markers can vary among populations, population-specific data are of high importance. AIM: To investigate the mutation rates of 16 STR markers in the Slovak population. SUBJECTS AND METHODS: In this study, we analysed the germline mutation rates of 16 STR markers (TH01, D3S1358, vWA, D21S11, D16S539, D1S1656, D19S433, SE33, D10S1248, D22S1045, D12S391, D8S1179, D2S1338, D2S441, D18S51 and FGA) in the Slovak population. At these loci, we analysed 42 096 allelic transfers and identified 61 mutation events. RESULTS: The loci with the highest overall mutation rates were SE33 and FGA, while no mutations were identified in TH01, D19S433 and D22S1045. The average paternal mutation rate was higher than the maternal mutation rate. All but one mutation consisted of gains or losses of a single repeat unit and the overall mutation rate was estimated to be 1.45 x10-3 per meiosis. CONCLUSION: This study provides data which can be used to further strengthen the correct paternity index calculations and reliability of paternity testing in Slovakia.

Performance of using opposing homozygotes for paternity testing in Japanese Black cattle.

Genome-wide single nucleotide polymorphism (SNP) markers in Japanese Black cattle enable genomic prediction and verifying parent-offspring relationships. We assessed the performance of opposing homozygotes (OH) for paternity testing in Japanese Black cattle, using SNP genotype information of 50 sires and 3,420 fattened animals, 1,945 of which were fathered by the 50 genotyped sires. The number of OH was counted for each sire-progeny pair in 28,764 SNPs with minor allele frequencies of ≥0.05 in this population. Across all pairs of animals, the number of OH tended to increase as the pedigree-based coefficient of relationship decreased. With a threshold of 288 (1% of SNPs) for paternity testing, most sire-progeny pairs were detected as true relationships. The frequency of Mendelian inconsistencies was 2.4%, reflecting the high accuracy of pedigree information in Japanese Black cattle population. The results indicate the utility of OH for paternity testing in Japanese Black cattle.

Genetic polymorphisms of 66 InDel loci in the Chinese Han population.

Insertion/deletion polymorphism (InDel) genetic markers refer to insertion or deletion of DNA fragments into genomic DNA, which have advantages in the identification of degraded samples. In this study, we independently screened 66 highly polymorphic InDel markers from the dbSNP database to establish a multiplex PCR system for forensic DNA identification using next-generation sequencing system (66-plex InDels). We assessed the population genetic data among 251 Chinese Han population using this system and evaluated their potential forensic application. The results showed that all 66 InDel loci conformed to the Hardy-Weinberg equilibrium (P>0.000 758), and all the pairwise InDel loci were in linkage equilibrium after Bonferroni correction. The mean observed heterozygosity (Ho) was 0.482, the mean expected heterozygosity (He) was 0.483,the mean discrimination power (DP) was 0.612, the mean polymorphism information content (PIC) was 0.365, the total discrimination power (TDP) reached 0.999 999 999 999 999 999 999 999 999 428 18. The cumulative power of exclusion for 66 InDel loci was 0.999 739 in duo cases (CPEduo) and was 0.999 999 999 417 in trios cases (CPEtrio). The results show that the 66 InDel loci have high genetic polymorphisms in the Chinese Han population and can be used independently for forensic DNA identification and paternity testing.

Detection of cell-free fetal DNA in maternal plasma using two types of compound markers.

With the discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing became possible. However, analysis of low-level cffDNA against high background maternal DNA remains complicated and challenging. To circumvent this limitation, selective amplification of cffDNA was used in this study. Two kinds of compound markers (namely DIP-STR and SNP-STR), both based on selective amplification, were used here for targeting fetal DNA. By designing two allele-specific forward primers for DIP-STR and SNP-STR, DNA fragments with different DIP/SNP alleles can be selectively amplified. When analyzing maternal plasma DNA, these markers can selectively target paternally inherited fetal alleles whose DIP/SNP allele was not shared with the mother. In this study, 21 families were studied with six DIP-STRs and 11 SNP-STRs. Fetal DNA was successfully detected across plasma samples for at least one marker. Detection rate varied between DIP-STR and SNP-STR markers, and DIP-STR outperforms SNP-STR. Fetal alleles obtained from maternal plasma were double confirmed by genotyping paternal genomic DNA and fetal genomic DNA from amniocentesis. This study demonstrated that selective amplification strategy can be used to target cffDNA in maternal plasma, which will be a promising method for noninvasive prenatal paternity testing.

Selection and evaluation of bi-allelic autosomal SNP markers for paternity testing in Koreans.

Due to the advantages of single-nucleotide polymorphisms (SNPs) in forensic science, many forensic SNP panels have been developed. However, the existing SNP panels have a problem that they do not reflect allele frequencies in Koreans or the number of markers is not sufficient to perform paternity testing. Here, we filtered candidate SNPs from the Ansan-Ansung cohort data and selected 200 SNPs with high allele frequencies. To reduce the risk of false inclusion and false exclusion, we calculated likelihood ratios of alleged father-child pairs from simulated families when the alleged father is the true father, the close relative of the true father, and the random man. As a result, we estimated that 160 SNPs were needed to perform paternity testing. Furthermore, we performed validation using Twin-Family cohort data. When 160 selected SNPs were used to calculate the likelihood ratio, paternity and non-paternity were accurately distinguished. Our set of 160 SNPs could be useful for paternity testing in Koreans.

Development of a 19-plex short tandem repeat typing system for individual identification and parentage testing of horses (Equus caballus).

Individual identification of horses for pedigree verification and registration is important for the sustainable development of the horse industry. Horse individual identification and parentage tests commonly use the 17 short tandem repeats (STRs) recommended by the International Society for Animal Genetics (ISAG) and the locus LEX33. While many multiplex STR typing systems have been established for the horse, a sex determining marker is usually absent, and none of them can simultaneously detect all 17 ISAG recommended loci and the locus LEX33. Here, we present a 19-plex STR typing system that contains the 17 ISAG recommend loci, the locus LEX33 and amelogenin as sex determining loci. The results of our sensitivity, species specificity, stutter analysis and population data analysis, indicate that this system is a specific, sensitive, and robust tool for the identification of individuals, parentage testing and genetic research in the horse.

Genetic analysis of tri-allelic patterns at the CODIS STR loci.

In the routine of autosomal STR genotyping for forensic aims, tri-allelic patterns could be occasionally observed at a single locus in phenotypically normal individuals. Two predominant types of tri-allelic variants have been nominated. Uneven intensities of three alleles are normally considered as the Type 1 pattern, and balanced height of three alleles are considered as the Type 2 pattern. In this study, the prevalence of tri-allelic patterns at the CODIS STR loci was investigated in global populations based on previous reports. The frequencies of the Type 1 and Type 2 pattern manifest a correlation with the germline mutation rates at the CODIS STR loci. The irregular high frequencies of the Type 2 pattern at TPOX with low germline mutation rates could attribute to the stable inheritance of genomic rearrangement from ancestral origin. Furthermore, results from genetic pattern analysis show that only a single allele from STRs with the Type 1 pattern could be transmitted from parents to offsprings, while a single allele and a combination of two alleles from STRs with the Type 2 pattern present an equal opportunity of transmission from parents to offsprings. Altogether, these results provide a genetic portrait of STRs with tri-allelic patterns, which will help the genetic interpretation of tri-allelic patterns in forensic practice.

Noninvasive prenatal paternity testing by maternal plasma DNA sequencing in twin pregnancies.

SNPs, combined with massively parallel sequencing technology, have proven applicability in noninvasive prenatal paternity testing (NIPPT) for singleton pregnancies in our previous research, using circulating cell-free DNA in maternal plasma. However, the feasibility of NIPPT in twin pregnancies has remained uncertain. As a pilot study, we developed a practical method to noninvasively determine the paternity of twin pregnancies by maternal plasma DNA sequencing based on a massively parallel sequencing platform. Blood samples were collected from 15 pregnant women (twin pregnancies at 9-18 weeks of gestation). Parental DNA and maternal plasma cell-free DNA were analyzed with custom-designed probes covering 5226 polymorphic SNP loci. A mathematical model for data interpretation was established, including the zygosity determination and paternity index calculations. Each plasma sample was independently tested against the alleged father and 90 unrelated males. As a result, the zygosity in each twin case was correctly determined, prior to paternity analysis. Further, the correct biological father was successfully identified, and the paternity of all 90 unrelated males was excluded in each case. Our study demonstrates that NIPPT can be performed for twin pregnancies. This finding may contribute to development in NIPPT and diagnosis of certain genetic diseases.

Preimplantation genetic testing as a component of root cause analysis of errors and reassignment of embryos in IVF.

The risks of embryo/gamete mix-up are a threat to the integrity of the IVF process, with significant implications for affected families. The use of preimplantation genetic testing through single-nucleotide polymorphism array or next-generation sequencing technology can help to identify, characterize and ultimately help, in some cases, to find the root cause, and to mitigate the extent of these errors for a given patient or laboratory.

A four-step mutation at D22S1045 in one complex paternity case when the brother of the alleged father hypothesis is evaluated.

We report one complex paternity case presenting a presumable paternal four-step STR mutation between the alleged father (AF) and child; the complexity of the case required the AF-brother hypothesis to be discarded without including this DNA sample. A total of 23 autosomal STR loci included in the Powerplex Fusion® and Globalfiler™ kits confirmed one isolated mismatch for D22S1045 between the AF (17/17) and the male child (13/15) in the presence of the mother (15/15). In this case, the STR structure and father's age do not seem to have contributed to promote the observed multistep mutation. The Paternity Index (PI) based on 23 autosomal STRs did not favor the AF paternity over the AF-brother hypothesis based on a flat prior (PI = 0.1217; W = 10.85%). For that reason, we included 38 autosomal human identification (HID) insertions-deletions (indels) and 20 retrotransposon insertion polymorphisms (RIPs) contained in the InnoTyper® 21 kit. Although these biallelic markers favored the AF paternity rather than the AF-brother hypothesis (LR = 110.3; W = 99.1%), the global PI based on 81 autosomal markers supported moderately the AF paternity hypothesis (LR = 13.4; W = 93.1%). The application of different mutation models showed a consistent support to the AF paternity hypothesis (PI = 93.1-99.95%), which could be useful for interpretation in these multistep STR mutation cases. In brief, we showed the impact of a four-step mutation at D22S1045 to obtain definitive paternity conclusions, particularly under a complex scenario when the AF-brother hypothesis is assessed. Forensic genomics arises as the next option for similar complex paternity cases.

Multiple methods used for type detection of uniparental disomy in paternity testing.

Uniparental disomy (UPD) has attracted more attention recently in paternity testing, though it is an infrequent genetic event. Although short tandem repeat (STR) profiling has been widely used in paternity testing, it is not sufficient to use STR only to judge the genetic relationship, because the existence of UPD will inevitably affect the results of genotyping. Compared with complete UPD, segmental UPD is more difficult to detect because it does not affect all genotypes on the same chromosome. It is necessary to determine the type of UPD with multiple methods because a single method is not sufficient. Therefore, it is advisable to detect UPD in paternity testing with multiple methods. In this study, after autosomal STR profiling was used, we found that there were several gene loci on the same chromosome that did not conform to Mendelian genetic law, thus we highly suspected the existence of UPD and performed X-STR profiling immediately. Then whole-genome single nucleotide polymorphism (SNP) array analysis was performed to identify the type, and the results provided straightforward evidence for distinguishing complete from segmental UPD. Lastly, we used deletion insertion polymorphism (DIP)-SNP SNaPshot assay and Miseq FGx sequencing (for SNP and STR) to determine whether the mutation source is maternal uniparental disomy (mUPD) or paternal uniparental disomy (pUPD). To avoid false exclusion of kinship, it is vital to determine the type of UPD in paternity testing and effective strategies based on multiple methods to detect the type of UPD are provided in this study.

Analysis of fetal DNA in maternal plasma with markers designed for forensic DNA mixture resolution.

PURPOSE: With the description of circulating fetal DNA in maternal blood, noninvasive prenatal diagnostics became theoretically possible. As the presence of background maternal DNA interferes with the detection of fetal DNA, analytical methods require genetic markers capable of distinguishing by quantitative or targeted approaches the minor population of DNA molecules of the fetus. Here we evaluate the feasibility of analyzing fetal DNA with novel DIP-STR genetic markers, designed for the investigation of forensic mixed biological evidence. METHODS: The DIP-STR molecular approach is based on sequence-specific analysis of paternally inherited fetal alleles. These sequences are biallelic deletion/insertion polymorphisms (DIPs) located very close to short tandem repeat (STR) markers, for combined analysis. In this study, 48 women were tested with 28 DIP-STRs during the first, second, and third trimester of pregnancy. RESULTS: Positive results were obtained across markers, including longer ones (386 base-pairs) and with blood samples collected during early pregnancy, such as 10 weeks of gestational age. CONCLUSION: These data show that DIP-STR markers can be used to amplify specific genomic regions of circulating fetal DNA to obtain targeted genetic information. This method may contribute to developments in noninvasive prenatal paternity testing and diagnosis of certain genetic diseases.

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 ).