Identifying the most likely contributors to a Y-STR mixture using the discrete Laplace method

In some crime cases, the male part of the DNA in a stain can only be analysed using Y chromosomal markers, e.g. Y-STRs. This may be the case in e.g. rape cases, where the male components can only be detected as Y-STR profiles, because the fraction of male DNA is much smaller than that of female DNA, which can mask the male results when autosomal STRs are investigated. Sometimes, mixtures of Y-STRs are observed, e.g. in rape cases with multiple offenders. In such cases, Y-STR mixture analysis is required, e.g. by mixture deconvolution, to deduce the most likely DNA profiles from the contributors.We demonstrate how the discrete Laplace method can be used to separate a two person Y-STR mixture, where the Y-STR profiles of the true contributors are not present in the reference dataset, which is often the case for Y-STR profiles in real case work. We also briefly discuss how to calculate the weight of the evidence using the likelihood ratio principle when a suspect's Y-STR profile fits into a two person mixture. We used three datasets with between 7 and 21 Y-STR loci: Denmark (n = 181), Somalia (n = 201) and Germany (n = 3443). The Danish dataset with 21 loci was truncated to 15 and 10 loci to examine the effect of the number of loci. For each of these datasets, an out of sample simulation study was performed: A total of 550 mixtures were composed by randomly sampling two haplotypes, h₁ and h₂, from the dataset.We then used the discrete Laplace method on the remaining data (excluding h₁ and h₂) to rank the contributor pairs by the product of the contributors’ estimated haplotype frequencies. Successful separation of mixtures (defined by the observation that the true contributor pair was among the 10 most likely contributor pairs) was found in 42–52% of the cases for 21 loci, 69–75% for 15 loci and 92–99% for 10 loci or less depending on the dataset and how the discrete Laplace model was chosen. Y-STR mixtures with many loci are difficult to separate, but even haplotypes with 21 Y-STR loci can be separated.     

A forensic DNA profiling system for Northern European brown bears (Ursus arctos)

A set of 13 dinucleotide STR loci (G1A, G10B, G1D, G10L, MU05, MU09, MU10, MU15, MU23, MU26, MU50, MU51, MU59) were selected as candidate markers for a DNA forensic profiling system for Northern European brown bear (Ursus arctos). We present results from validation of the markers with respect to their sensitivity, species specificity and performance (precision, heterozygote balance and stutter ratios). All STRs were amplified with 0.6 ng template input, and there were no false bear genotypes in the cross-species amplification tests. The validation experiments showed that stutter ratios and heterozygote balance was more pronounced than in the tetranucleotide loci used in human forensics. The elevated ratios of stutter and heterozygote balance at the loci validated indicate that these dinucleotide STRs are not well suited for interpretation of individual genotypes in mixtures. Based on the results from the experimental validations we discuss the challenges related to genotyping dinucleotide STRs in single source samples. Sequence studies of common alleles showed that, in general, the size variation of alleles corresponded with the variation in number of repeats. The samples characterized by sequence analysis may serve as standard DNA samples for inter laboratory calibration. A total of 479 individuals from eight Northern European brown bear populations were analyzed in the 13 candidate STRs. Locus MU26 was excluded as a putative forensic marker after revealing large deviations from expected heterozygosity likely to be caused by null-alleles at this locus. The remaining STRs did not reveal significant deviations from Hardy–Weinberg equilibrium expectations except for loci G10B and MU10 that showed significant deviations in one population each, respectively. There were 9 pairwise locus comparisons that showed significant deviation from linkage equilibrium in one or two out of the eight populations. Substantial genetic differentiation was detected in some of the pairwise population comparisons and the average estimate of population substructure (F_ST) was 0.09. The average estimate of inbreeding (F_IS) was 0.005. Accounting for population substructure and inbreeding the total average probability of identity in each of the eight populations was lower than 1.1 × 10^?9 and the total average probability of sibling identity was lower than 1.3 × 10^?4. The magnitude of these measurements indicates that if applying these twelve STRs in a DNA profiling system this would provide individual specific evidence.     

Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples

Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR amplification of tandem repeats. These artifacts may be avoided by typing bi-allelic markers instead of STRs. In this work, the SNPforID multiplex assay was used to type LtDNA. A sensitized SNP typing protocol was introduced, that increased signal strengths without increasing noise and without affecting the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two different consensus models were used to interpret the SNP data. Correct profiles with 42–49 SNPs were generated from the 50 and 100 pg experiments, whereas a few incorrect genotypes were included in the generated profiles from the 25 pg experiments. With the strict consensus model, between 35 and 48 SNPs were correctly typed in the 25 pg experiments and only one allele drop-out (error rate: 0.07%) was observed in the consensus profiles.A total of 28 crime case samples were selected for typing with the sensitized SNPforID protocol. The samples were previously typed with old STR kits during the crime case investigation and only partial profiles (0–6 STRs) were obtained. Eleven of the samples could not be quantified with the Quantifiler™ Human DNA Quantification kit because of partial or complete inhibition of the PCR. For eight of these samples, SNP typing was only possible when the buffer and DNA polymerase used in the original protocol was replaced with the AmpFℓSTR^® SEfiler Plus™ Master Mix, which was developed specifically for challenging forensic samples. All the crime case samples were successfully typed with the SNPforID multiplex assay and the match probabilities ranged from 1.1 × 10⁻¹⁵ to 7.9 × 10⁻²³. In comparison, four of the samples could not be typed with the AmpFℓSTR^® SEfiler Plus™ kit and the match probabilities were higher than 10⁻⁷ for another six samples.     

Developmental and internal validation of a novel 13 loci STR multiplex method for Cannabis sativa DNA profiling

Marijuana (Cannabis sativa L.) is a plant cultivated and trafficked worldwide as a source of fiber (hemp), medicine, and intoxicant. The development of a validated method using molecular techniques such as short tandem repeats (STRs) could serve as an intelligence tool to link multiple cases by means of genetic individualization or association of cannabis samples. For this purpose, a 13 loci STR multiplex method was developed, optimized, and validated according to relevant ISFG and SWGDAM guidelines. The STR multiplex consists of 13 previously described C. sativa STR loci: ANUCS501, 9269, 4910, 5159, ANUCS305, 9043, B05, 1528, 3735, CS1, D02, C11, and H06. A sequenced allelic ladder consisting of 56 alleles was designed to accurately genotype 101 C. sativa samples from three seizures provided by a U.S. Customs and Border Protection crime lab. Using an optimal range of DNA (0.5–1.0 ng), validation studies revealed well-balanced electropherograms (inter-locus balance range: 0.500–1.296), relatively balanced heterozygous peaks (mean peak height ratio of 0.83 across all loci) with minimal artifacts and stutter ratio (mean stutter of 0.021 across all loci). This multi-locus system is relatively sensitive (0.13 ng of template DNA) with a combined power of discrimination of 1 in 55 million. The 13 STR panel was found to be species specific for C. sativa; however, non-specific peaks were produced with Humulus lupulus. The results of this research demonstrate the robustness and applicability of this 13 loci STR system for forensic DNA profiling of marijuana samples.     

Expanding beyond the current core STR loci: An exploration of 73 STR markers with increased diversity for enhanced DNA mixture deconvolution

Current approaches to mixture deconvolution of complex biological samples at times do not have the capability to resolve component contributors in DNA evidence. Additional short tandem repeat (STR) loci were sought that may improve the forensic genetic analysis of mixtures. This study presents exploratory data of a multiplex comprised of 73 highly polymorphic STRs (referred herein as the 73Plex) that were selected because of their high diversity due to sequence variation. These STRs (or a subset of them) may be considered as candidates that may augment current core markers capabilities for DNA mixture deconvolution. Population genetics analyses were performed for each locus using DNA samples from 451 individuals comprising three U.S. populations. Sequence-based heterozygosities ranged from 72% to 98%, where only two loci (D10A97 and D6A7) fell below 80%. Mixture deconvolution capabilities for two-person mixtures were assessed based on complete allele resolution per locus (i.e., four alleles observed) of pairwise mixtures using in silico methods. A subset of 20 highly informative loci (referred herein as the 20Plex) from the 73Plex was compared to the 20 CODIS core loci on all population samples with full DNA profiles for both panels (i.e., no locus dropout; n = 443). Based on proportion of loci displaying four alleles, the 20Plex outperformed the CODIS core loci with increases of 82.6% and 89.3% using length-based and sequence-based alleles, respectively. A combination of 17 STR from the 20Plex and 3 CODIS loci gave the highest capacity for resolving allelic components per locus. These data illustrate the increased value of utilizing sequenced-based alleles of additional STR loci. Furthermore, there are a number of candidate STR loci that could notably augment the current core STR loci and enhance mixture interpretation capabilities.     

Korean population genetic data and concordance for the PowerPlex® ESX 17, AmpFlSTR Identifiler®, and PowerPlex® 16 systems

In case of paternity or maternity investigations with short tandem repeat (STR) analysis, deficient cases, missing person, or mutations are encountered and common STRs cannot provide sufficient forensic parameters. Thus, it is recommended that additional STRs are needed to complement conventional analysis for more reliable forensic information. We analyzed variation of 23 STRs contained in the new PowerPlex^® ESX 17 kit (Promega) and two conventional kits of the AmpFlSTR Identifiler^® (Applied Biosystems) and PowerPlex^® 16 systems (Promega) in 452 randomly chosen individuals from Korea to provide an expanded and reliable Korean database. Allele frequencies and forensic parameters were used to evaluate suitability and robustness of the new kit for forensic genetic analysis as well as in concordance studies. The combined probability of match for the 16 loci in the PowerPlex^® ESX 17 system was 2.76 × 10⁻²⁰. One genotyping discrepancy due to a null allele was observed at the D18S51 locus (the concordant rate = 99.99%), showing a primer-binding site mutation in the sequence of the locus (G-to-A substitution at position 146 of Genbank accession number JX018211). Thus, the new kit is a valuable forensic tool and is suitable to extend the Korean population genetic data obtained with well-established polymerase chain reaction multiplex-kits of the AmpFlSTR Identifiler^® and PowerPlex^® 16 systems.     

Altered spontaneous brain activity pattern in cognitively normal young adults carrying mutations of APP,presenilin-1/2 and APOE ε4

ObjectiveTo explore genetic effects of amyloid precursor protein (APP), presenilin-1/2 and apolipoprotein E (APOE) ε4 on brain structural and functional alterations in cognitively normal young adults.Materials and methodsEighty healthy adults (mean age 24.0 ± 2.5 years; n = 18, APP/presenilin-1/2 group; n = 31, APOE ε4 group; n = 31, control group [without above-mentioned gene mutation]) underwent high-resolution T1-weighted 3D anatomical imaging, resting-state functional MR imaging and neuropsychological assessments. We used voxel-based morphometry and regional homogeneity (ReHo) algorithms to investigate brain structural and functional changes among three groups, and performed correlation analyses between the brain regions with statistically significant difference and neuropsychological results.ResultsNo brain structural changes were found, however, ReHo values were increased in right parietal-frontal lobes in APOE ε4 group, and decreased in the left middle temporal gyrus in APP/presenilin-1/2 group compared with controls (all P < 0.05). Compared with APOE ε4 group, decreased ReHo values of bilateral temporal lobes were shown in APP/presenilin-1/2 group (P < 0.05). ReHo values of right superior frontal gyrus in APOE ε4 group positively correlated with neuropsychological tests scores(P < 0.05).ConclusionCognitively normal young adults carrying APOE ε4 or APP/presenilin-1/2 had different spontaneous brain activity patterns without cerebral structural differences.     

European Network of Forensic Science Institutes (ENFSI): Evaluation of new commercial STR multiplexes that include the European Standard Set (ESS) of markers

To support and to underpin the European initiative to increase the European set of standard markers (ESS), by the addition of five new loci, a collaborative project was organised by the European Network of Forensic Science Institutes (ENFSI) DNA working group in order to assess the new multiplex kits available. We have prepared allele frequency databases from 26 EU populations. Concordance studies were carried out to verify that genotyping results were consistent between kits. Population genetics studies were conducted and it was estimated that F_ST < 0.001. The results showed that the kits were comparable to each other in terms of performance and major discrepancy issues were highlighted. We provide details of allele frequencies for each of the populations analysed per laboratory.     

Evaluation of forensic examination of extremely aged seminal stains

The results of forensic tests, such as semen identification and short tandem repeat (STR) analysis of extremely aged seminal stains from unsolved sex crimes can provide important evidence. In this study we evaluated whether current forensic methods could be applied to seminal stains that were stored at room temperature for 33–56 years (n = 2, 33 years old; n = 1, 41 years old; n = 1, 44 years old; n = 1, 56 years old). The prostatic acid phosphatase (SM-test reagent), microscopic (Baecchi stain method) and semenogelin (RSID™ Semen Laboratory Kit) tests were performed as discriminative tests for semen. In addition, the mRNA levels of the semen-specific proteins semenogelin 1 (SEMG1) and protamine 2 (PRM2) were investigated. STRs were analyzed using the AmpFlSTR® Identifiler™ PCR Amplification Kit. All samples were positive in the prostatic acid phosphatase and semenogelin tests, and sperm heads were identified in all samples. The staining degree of the aged sperm heads was similar to that of fresh sperm. Although SEMG1 mRNA was not detected in any sample, PRM2 mRNA was detected in three samples. In the STR analysis, all loci were detected in the 33-years-old sample and five loci were detected in the 56-years-old sample. We confirmed that current forensic examinations – including STR analysis – could be applied to extremely aged seminal stains. These results could be useful for forensic practice.     

Alcohol and drugs in suspected impaired drivers in Ontario from 2001 to 2005

IntroductionBlood samples from 733 drivers suspected of driving under the influence of alcohol in the province of Ontario from 2001 to 2005 were retrospectively examined.MethodsSamples were analyzed for alcohol content by headspace gas chromatography with flame ionization detection. Drivers ranged in age from 15 to 83 years old with the majority of blood samples obtained from males (n = 623, 85%). Of the 704 cases where quantifiable numerical values were obtained, blood alcohol concentrations ranged from 13 to 414 mg/100 mL (mean 172 mg/100 mL) for males and 10 to 425 mg/100 mL (mean 173 mg/100 mL) for females. The majority of these drivers (n = 640/704, 90.9%) had blood alcohol concentrations of 80 mg/100 mL and greater at the time of sampling. Analysis for alcohol was undertaken in all cases. However, additional toxicological examinations for drugs was conducted on a case-by-case basis based on the submitted case history and/or where there were requests for additional drug analysis, or where such analysis would be probative in the absence of the detection of alcohol at a concentration that could cause impairment.ResultsTherefore, analyses for drugs were only performed in a small subset of 42 cases (6%). Thirty-four of these cases had positive drug findings, with Δ⁹-tetrahydrocannabinol being the most frequently encountered drug (n = 18), followed by benzoylecgonine/cocaine (n = 8), morphine (n = 6), lorazepam (n = 5) and diphenhydramine (n = 4). The majority of individuals were involved in some type of motor vehicle accident (n = 658, 89.8%), with single motor vehicle accidents (n = 412, 56.2%) being the most common, followed by multiple motor vehicle accidents (n = 169, 23%). Injuries (n = 309, 42.1%) were the main cause of individuals not being able to provide breath samples with specific, non-life threatening injuries (n = 178, 24.3%) representing the highest percentage of cases. The majority of incidents (n = 449, 61.3%) occurred between Friday and Sunday reaching a peak on Saturday (n = 174, 23.7%). Incidents occurred throughout the day, with the majority of events (n = 449/705, 63.7%) for which a time was provided occurring between 6:01 pm and 3:00 am, and the peak number of incidents occurring between 9:01 pm and midnight (n = 168/705, 23.8%).ConclusionHowever, these data demonstrate that ‘‘drugged driving” does occur and that further, comprehensive investigation is needed to determine the frequency and type of drug use by Ontario drivers.     

A novel set of DIP-STR markers for improved analysis of challenging DNA mixtures

Currently available molecular biology tools allow forensic scientists to characterize DNA evidence found at crime scenes for a large variety of samples, including those of limited quantity and quality, and achieve high levels of individualization. Yet, standard forensic markers provide limited or no results when applied to mixed DNA samples where the contributors are present in very different proportions (unbalanced DNA mixtures). This becomes an issue mostly for the analysis of trace samples collected on the victim or from touched objects.To this end, we recently proposed an innovative type of genetic marker, named DIP-STR that relies on pairing deletion/insertion polymorphisms (DIP) with standard short tandem repeats (STR). This novel compound marker allows detection of the minor DNA contributor in a DNA mixture of any gender and cellular origin with unprecedented resolution (beyond a DNA ratio of 1:1000).To provide a novel analytical tool useful in practice to common forensic laboratories, this article describes the first set of 10 DIP-STR markers selected according to forensic technical standards. The novel DIP-STR regions are short (between 146 and 271 bp), include only highly polymorphic tri-, tetra- and pentanucleotide tandem repeats and are located on different chromosomes or chromosomal arms to provide statistically independent results. This novel set of DIP-STR can target the amplification of 0.03–0.1 ng of DNA when mixed with a 1000-fold excess of major DNA. DIP-STR relative allele frequencies are estimated based on a survey of 103 Swiss individuals. Finally, this study provides an estimate of the occurrence of informative alleles and a calculation of the corresponding random match probability of the detected minor DIP-STR genotype assessed across 10,506 pairwise conceptual mixtures.     

A SNaPshot of next generation sequencing for forensic SNP analysis

Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot^®, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30–40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies’ Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.     

Open source software EuroForMix can be used to analyse complex SNP mixtures

A series of two- and three-person mixtures of varying dilutions were prepared and analysed with Life Technologies’ HID-Ion AmpliSeq™ Identity Panel v2.2 using the Ion PGM™ massively parallel sequencing (MPS) system. From this panel we used 134 autosomal SNPs. Using the reference samples of three donors, we evaluated the strength of evidence with likelihood ratio (LR) calculations using the open-source quantitative EuroForMix program and compared the results with a previous study using a qualitative software (LRmix). SNP analysis is a special case of STRs, restricted to a maximum of two alleles per locus. We showed that simple two-person mixtures can be readily analysed with both LRmix and Euroformix, but the performance of three- or more person mixtures is generally inefficient with LRmix. Taking account of the “peak height” information, by substituting ‘sequence read’ coverage values from the MPS data for each SNP allele, greatly improves the discrimination between true and non-contributors. The higher the mixture proportion (Mx) of the person of interest is, the higher the LR. Simulation experiments (up to six contributors) showed that the strength of the evidence is dependent upon Mx, but relatively insensitive to the number of contributors. If a higher number of loci were multiplexed, the analysis of mixtures would be much improved, because the extra information would enable lower Mx values to be evaluated. In summary, incorporating the 'sequence read' (coverage) into the quantitative model shows a significant benefit over the qualitative approach. Calculations are quite fast (six seconds for three contributors).     

Massively parallel sequencing analysis of nondegraded and degraded DNA mixtures using the ForenSeq™ system in combination with EuroForMix software

Massively parallel sequencing (MPS) technologies enable the simultaneous analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). MPS also enables the detection of alleles of the minor contributors in imbalanced DNA mixtures. In this study, 59 STRs (amelogenin, 27 autosomal STRs, 7 X-STRs, and 24 Y-STRs) and 94 identity-informative SNPs of 119 unrelated Taiwanese (50 men, 69 women) were sequenced using a commercial MPS kit. Forty-eight nondegraded and 44 highly degraded two-person artificial DNA mixtures with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, and 1:99) were analyzed to examine the performance of this system for detecting the alleles of the minor contributors in DNA mixtures. Likelihood ratios based on continuous model were calculated using the EuroForMix for DNA mixture interpretation. The STR and SNP genotypes of these 119 Taiwanese were obtained. Several sequence variants of STRs were observed. Using EuroForMix software based on the sequence data of autosomal STRs and autosomal SNPs, 97.9% (47/48) and 97.7% (42/43) of minor donors were accurately inferred among the successfully analyzed nondegraded and degraded DNA mixtures, respectively. In conclusion, combined with EuroForMix software, this commercial kit is effective for assignment of the minor contributors in nondegraded and degraded DNA mixtures.

     

Population genetic evaluation of eight X-chromosomal short tandem repeat loci using Mentype Argus X-8 PCR amplification kit

The evaluation of four pairs of X-chromosomal short tandem repeats (STRs), i.e. DXS10135–DXS8378, DXS7132–DXS10074, HPRTB-DXS10101 and DXS7423–DXS10134 was carried out using the Argus X-8 Multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome (ChrX), and for practical reasons they are assigned to four linkage groups 1–4. The genetic distance within the STR pairs is assumed to be <1 cM, whereas the pair to pair space is about 50 cM or more. Here, we present single STR allele frequencies, haplotype frequencies of the respective STR pairs and further population genetic parameters of forensic interest. Most data refer to a German population, however small samples from Ghana and Japan were also investigated. Furthermore, sequencing of all STR loci displayed the presence of microvariant alleles and variations in the repeat flanking region. A total of 350 meioses investigated here revealed only one sperm DXS7132 mutation. For analysis of linkages within the STR pairs a study involving 104 female meiosis with respect to recombination events was performed. The STR panel presented here provides a powerful tool for solving complex kinship in the case that X-chromosomal lineages can be taken under investigation.     

DNA methylation-based age prediction from saliva: High age predictability by combination of 7 CpG markers

DNA methylation is currently one of the most promising age-predictive biomarkers. Many studies have reported DNA methylation-based age predictive models, but most of these are based on DNA methylation patterns from blood. Only a few studies have examined age-predictive DNA patterns in saliva, which is one of the most frequently-encountered body fluids at crime scenes. In this study, we generated genome-wide DNA methylation profiles of saliva from 54 individuals and identified CpG markers that showed a high correlation between methylation and age. Because the age-associated marker candidates from saliva differed from those of blood, we investigated DNA methylation patterns of 6 age-associated CpG marker candidates (cg00481951, cg19671120, cg14361627, cg08928145, cg12757011, and cg07547549 of the SST, CNGA3, KLF14, TSSK6, TBR1, and SLC12A5 genes, respectively) in addition to a cell type-specific CpG marker (cg18384097 of the PTPN7 gene) in an independent set of saliva samples obtained from 226 individuals aged 18 to 65 years. Multiplex methylation SNaPshot reactions were used to generate the data. We then generated a linear regression model with age information and the methylation profile from the 113 training samples. The model exhibited a 94.5% correlation between predicted and chronological age with a mean absolute deviation (MAD) from chronological age of 3.13 years. In subsequent validation using 113 test samples, we also observed a high correlation between predicted and chronological age (Spearman’s rho = 0.952, MAD from chronological age = 3.15 years). The model composed of 7 selected CpG sites enabled age prediction in saliva with high accuracy, which will be useful in saliva analysis for investigative leads.     

Complex mixtures: a critical examination of a paper by Homer et al

DNA evidence in criminal cases may be challenging to interpret if several individuals have contributed to a DNA-mixture. The genetic markers conventionally used for forensic applications may be insufficient to resolve cases where there is a small fraction of DNA (say less than 10%) from some contributors or where there are several (say more than 4) contributors. Recently methods have been proposed that claim to substantially improve on existing approaches. The basic idea is to use high-density single nucleotide polymorphism (SNP) genotyping arrays including as many as 500,000 markers or more and explicitly exploit raw allele intensity measures. It is claimed that trace fractions of less than 0.1% can be reliably detected in mixtures with a large number of contributors. Specific forensic issues pertaining to the amount and quality of DNA are not discussed in the paper and will not be addressed here. Rather our paper critically examines the statistical methods and the validity of the conclusions drawn in Homer et al. (2008). We provide a mathematical argument showing that the suggested statistical approach will give misleading results for important cases. For instance, for a two person mixture an individual contributing less than 33% is expected to be declared a non-contributor. The quoted threshold 33% applies when all relative allele frequencies are 0.5. Simulations confirmed the mathematical findings and also provide results for more complex cases. We specified several scenarios for the number of contributors, the mixing proportions and allele frequencies and simulated as many as 500,000 SNPs. A controlled, blinded experiment was performed using the Illumina GoldenGate(?) 360 SNP test panel. Twenty-five mixtures were created from 2 to 5 contributors with proportions ranging from 0.01 to 0.99. The findings were consistent with the mathematical result and the simulations. We conclude that it is not possible to reliably infer the presence of minor contributors to mixtures following the approach suggested in Homer et al. (2008). The basic problem is that the method fails to account for mixing proportions.     

TPH1 A218 allele is associated with suicidal behavior in Turkish population

BackgroundSerotonergic dysfunction is implicated in depression, psychiatric disorders and suicidal behaviors. The first and rate-limiting step in the synthesis of serotonin is catalyzed by tryptophan hydroxylase (TPH) which is encoded by TPH1 and THP2 genes. Genetic association studies have revealed contradictory results about the effect of the TPH1 A218C (rs1800532) polymorphism on suicidal behavior in different populations.Material and methodIn this study, we investigated A218C polymorphism in 109 suicide attempters and 98 healthy controls. Socio-demographic characteristics of participants were obtained through questionnaire. DNA was extracted from peripheral blood and genotyping was performed by Real Time PCR. Fisher’s exact test was used to evaluate the significance of the difference among the independent variables. Hardy-Weinberg equilibrium was tested using Pearson’s goodness-of-fit chi-squared test.ResultsThe frequency of A allele was significantly higher in suicide attempters than controls (46.33% vs. 35.71%, p = 0.0357). However, there were no differences in genotype frequencies of this locus between participants having attempted suicide and controls (p > 0.05). Among males, frequencies of CC genotype and C allele were found to be significantly higher in controls (p = 0.0125, p = 0.0298). With regard to the female subjects and female controls, no significant association was detected between suicidal behavior and genotype/allele frequencies (p > 0.05).ConclusionOur results provide evidence that A allele of TPH1 A218C polymorphism may be associated with suicidal behavior in Turkish population.     

Analysis of nondegraded and degraded DNA mixtures of close relatives using massively parallel sequencing

Identification of the minor contributor in DNA mixture of close relatives remains a dilemma in forensic genetics. Massively parallel sequencing (MPS) can analyze multiple short tandem repeats (STRs) and single nucleotide polymorphism (SNPs) concurrently and detect non-overlapping alleles of the minor contributors in DNA mixtures. A commercial kit for MPS of 59 identity informative STRs (iiSTRs) and 94 autosomal identity-informative SNPs (iiSNPs) was used to analyzed 34 nondegraded and 33 highly degraded two-person artificial DNA mixtures of close relatives with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, 1:99). EuroForMix software was used to determine the minor contributors in the mixtures based on the likelihood ratios calculated from the MPS data, and relMix software was used to perform kinship analysis of the contributors. The STRs and SNPs of the 34 nondegraded and 33 degraded DNA mixtures were genotyped using MPS. Using EuroForMix based on the genotypes of autosomal iiSTRs and autosomal iiSNPs, 82.4% (28/34) and 54.5% (18/33) of minor donors could be accurately assigned for the nondegraded and degraded DNA mixtures, respectively. The relMix software correctly inferred the relationship between contributors in 97.1% (33/34) of nondegraded mixtures and in 97.0% (32/33) of degraded mixtures. In conclusion, combined EuroForMix and MPS data of STRs and SNPs can assist in the assignment of minor donors in nondegraded DNA mixtures of close relatives, and relMix can be used to infer relationship among contributors.