SNPs in paternity investigation: The simple future

Based on the 52 SNP-plex developed by the SNPforID Consortium, we designed two 10-plex to study single nucleotide polymorphisms (SNPs) for human identification and to establish its usefulness in paternity casework. This 20 autosomal SNP set was studied in 56 paternity investigation cases from South Portuguese resident population, also analyzed with 17 Short Tandem Repeats (STRs). Results obtained with both methodologies were consistent with each other, except for one case where the alleged father could not be excluded by SNPs. No mutation was found in the SNP loci, whereas a mismatch in STRs was detected. The use of SNPs as a complement to the analysis of autosomal STRs in paternity casework can result in paternity index and paternity probability values equivalent or higher than those obtained with more STR loci, but with lower costs. This study shows that instead of using additional STR loci, the analysis of 20 autosomal SNPs, as a complement technique to standard methodologies, is an appealing alternative in paternity investigation cases.     

Comprehensive analyses for genetic diversities of 19 autosomal STRs in Chinese Kazak group and its phylogenetic relationships with other continental populations

Short tandem repeats (STRs) play an essential role in forensic genetics due to their high degree of polymorphisms, wide distributions and easy detection method. In this study, allelic frequencies and forensic statistical parameters of the 19 autosomal STR loci in a Kazak ethnic group were calculated, and its genetic relationships with reference populations were assessed in order to understand population structure better and enrich population genetic data for forensic practice in Chinese Kazak ethnic group. There were 226 identified alleles with the corresponding allelic frequencies ranging from 0.0008 to 0.5295 in the 628 unrelated healthy Kazak individuals in Xinjiang Uygur Autonomous Region. All autosomal STRs were conformed to the Hardy-Weinberg equilibrium after Bonferroni’s correction. The cumulative power of discrimination and the combined probability of exclusion of all the 19 autosomal STRs were 0.999 999 999 999 999 999 999 997 162 and 0.999 999 994 484, respectively. Furthermore, the D_A distances and Fixation index values of pairwise populations, principal component analysis, multidimensional scaling analysis, phylogenetic tree analysis and structure analysis were conducted to probe the genetic relationships between the Kazak group and other reference populations. The population genetic results showed that these 19 autosomal STR loci were characterised by high genetic diversities in the Kazak group. Furthermore, the studied Kazak group had close genetic relationships with the Uyghur group and the Uzbek group. The present results may facilitate understanding the genetic background of the Chinese Xinjiang Kazak group.     

Determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction and capillary electrophoresis with PDA detection

A new method was developed for pre-concentration and determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction (DLLME) and capillary electrophoresis (CE) with photodiode array detection. The method was based on the formation of tiny droplets of an organic extractant in the prepared sample solution using water-immiscible organic solvent (chloroform) dissolved in water-miscible organic dispersive solvent (isopropyl alcohol). The organic phase, which extracted eight drugs of abuse from the prepared urine solution, was separated by centrifugation. The sedimented phase was transferred into a small volume CE auto-sampler vial with 10 µL of 1% HCl methanol solution and evaporated to dryness. The residue was reconstituted in lidocaine hydrochloride (internal standard) aqueous solution and introduced by electrokinetic injection into CE. Under the optimum conditions, acceptable linear relationship was observed in the range of 3.0–500 ng/mL with the correlation coefficient (r) of 0.9982–0.9994 for spiked urine samples. The limit of detection (LOD) (S/N = 3) was estimated to be 1.0 ng/mL. A recovery of 75.7%–90.6% was obtained for spiked samples. The mean relative error (MRE) was within ±7.0% and the relative standard deviation (RSD) was less than 6.9%. The proposed DLLME-CE procedure offers an alternative analytical approach for the sensitive detection of drugs of abuse in real urine samples.

Key points

• The dispersive liquid-liquid microextraction (DLLME) was involved for the determination of drugs in urine with capillary electrophoresis with photodiode array detection (CE-PDA).
• Good linearity, sensitivity, recovery and precision were achieved.
• The proposed method was eco-friendly with microliter scale solvent consumption.

     

Next generation sequencing technology in Second World War victim identification

The killings during the Second World War (WWII), with nearly 100,000 victims, is one of the greatest losses of life in Slovenia’s modern history and most of the victims are still buried in hidden mass graves and remain unidentified. Identity, ancestry, and phenotypic SNPs, as well as STR markers are already used for solving various cases with Next Generation Sequencing (NGS) technology. In this study, the Precision ID GlobalFiler NGS STR panel was used to identify the WWII victim that could not be identified with capillary electrophoresis (CE) analyses because limited statistical support was obtained after amplification of autosomal STRs using CE STR kits. Bones and teeth were analysed and compared to family references (nephew and niece on paternal line). Prior to DNA isolation 0.5 g of powder was decalcified. The DNA was purified in a Biorobot EZ1 device. The nuclear DNA of the samples was quantified with the PowerQuant kit. Because the recommended posterior probability (PP) of 99.9% was followed with the goal of high confidence of correct identification, the NGS STR Panel was used, and after the analysis of additional STR loci the statistical calculation showed a PP of 99.99986%, showing that a large enough number of genetic markers were analysed when identifying the skeletal remains of the aunt. PP value endorsed the hypothesis that the tooth and bone samples were from individual related to the family references rather than from unrelated individual. In presented case, NGS technology proved to be a powerful tool for increasing the number of autosomal STRs needed for identification of WWII victims when linear markers cannot be used for comparison and only distant relatives are available for analyses.     

Trisomy 21 disclosure using STR and SNP markers typed by MiSeq FGx™ Forensic Genomics System

The presence of a tri-allelic pattern at a single locus in a multiplex short tandem repeat (STR) profile is a rarely observable event. Generally, based on peak height measured by the capillary electrophoresis (CE) method and combination of alleles, the tri-allelic pattern is distinguishable into two predominant types: type 1 and 2, which are caused, respectively, by somatic mutations and chromosomal rearrangements. When tri-allelic patterns at more than one STR located on the same chromosome are detected, there is a reasonable suspicion of a trisomy due to an extra copy of a chromosome. Therefore, information on the type of three-band pattern is usually limited to STRs localized on the same chromosome included in the forensic kit in use and sometimes in insufficient numbers to classify this event correctly. The opportunity to extend this evaluation to additional markers, such as SNPs detectable using NGS, has not yet been explored. In this study, using the ForenSeq™ DNA Signature Prep kit, two cases of autosomal aneuploidy were revealed on chromosome 21, relying not only on STRs assessment but also extending the analysis to the five identity-informative single nucleotide polymorphisms (iiSNPs) localized on chromosome 21.     

Texas Population Substructure and Its Impact on Estimating the Rarity of Y STR Haplotypes from DNA Evidence*

Abstract: Three sampled populations of unrelated males—African American, Caucasian, and Hispanic, all from Texas—were typed for 16 Y short tandem repeat (STR) markers using the AmpFlSTR^® Yfiler^TM kit. These samples also were typed previously for the 13 core CODIS autosomal STR loci. Most of the 16 marker haplotypes (2478 out of 2551 distinct haplotypes) were observed only once in the data sets. Haplotype diversities were 99.88%, 99.89%, and 99.87% for the African American, Caucasian, and Hispanic sample populations, respectively. F_ST values were very small when a haplotype comprised 10–16 markers. This suggests that inclusion of substructure correction is not required. However, haplotypes consisting of fewer loci may require the inclusion of F_ST corrections. The testing of independence of autosomal and Y STRs supports the proposition that the frequencies of autosomal and Y STR profiles can be combined using the product rule.     

Application of Cameriere’s method for dental age estimation in children in South China

The aim of this study was to evaluate the applicability of Cameriere’s European formula for age estimation in children in South China and to adapt the formula to establish a more suitable formula for these children. Moreover, the performance of dental age estimation based on Cameriere’s method combining the developmental information of permanent teeth (PT) and third molar (TM) was also analysed. Orthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed. The samples of Group A were divided into training dataset 1 and test dataset 1, and the samples of Group B were also divided into training dataset 2 and test dataset 2. A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere’s European formula and the South China-specific formula was conducted with the test dataset 1. Additionally, a PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2. The Cameriere’s European formula underestimated chronological age with a mean difference (ME) of −0.47 ± 1.11 years in males and −0.69 ± 1.19 years in females. However, the South China-specific formula underestimated chronological age, with a mean difference (ME) of −0.02 ± 0.71 years in males and −0.14 ± 0.73 years in females. Compared with PT model and TM model, the PT and TM combined model obtained the smallest root mean square error (RMSE) of 1.29 years in males and 0.93 years in females. In conclusion, the South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children.

Key points

• Orthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed.
• A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere’s European formula and the South China-specific formula was conducted with the test dataset 1.
• A PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2.
• The South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children.

     

Allele frequencies of 15 STRs in the Calchaqui Valleys population (North-Western Argentina)

Allele frequencies for 15 short tandem repeat (STR) loci were obtained from a sample of 110 individuals from the Calchaqui Valleys population (North-Western Argentina). The combined power of exclusion and combined power of discriminating for the 15 tested STR loci were 0.999964 and 0.9999999999999998, respectively. Matching probability was 1 in 4.58 × 10(15). Therefore, it may be concluded that the set of 15 STRs included in the AmpF STR Identifiler kit, represents a powerful tool for forensic applications, paternity testing and population genetics studies in the Calchaqui Valleys population.     

Additional sequence characterization of NIST SRM 2391c: PCR-Based DNA Profiling Standard

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was designed for use in the standardization of forensic and paternity quality assurance procedures for fragment-based typing short tandem repeat (STR) alleles generated by the polymerase chain reaction (PCR). Certified genotypes of the 6 components A–F were assigned for 24 autosomal and 17 Y-STR markers plus Amelogenin using concordance testing between commercial kits. Selected Sanger sequencing characterization was performed for the alleles of 11 STR markers when only one PCR primer set was available for fragment-based typing. The goal is to characterize the remaining 30 STR loci in components A–C by Sanger sequencing methods for the STR repeat regions and adjacent flanking regions. Additional characterization of the SRM is intended to support the emerging interest in next-generation sequencing technologies for forensic typing applications. Sanger methods have detected underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. The sequenced regions include the commercial or known PCR binding sites commonly implemented in fragment-based typing.     

Developmental validation of 15 X chromosomal short tandem repeat markers

The use of X chromosomal short tandem repeat (STR) markers has been greatly increasing in the forensic setting. Using guidelines set forth previously for the validation of autosomal and Y STRs, aspects of the feasibility of routine X chromosomal STR use were evaluated. Two mini-X chromosomal STR multiplexes capable of amplifying 15 total markers were developed and utilized to determine allele nomenclature, allele/genotype frequencies, mutation rates, and linkage between markers. Additionally, a concordance study between these multiplexes and a commercially available kit was performed. Here, the authors present an overview of this extensive developmental validation study.     

Performance of the SNPforID 52 SNP-plex assay in paternity testing

The performance of a multiplex assay with 52 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was tested on 124 mother–child–father trios. The typical paternity indices (PIs) were 10⁵–10⁶ for the trios and 10³–10⁴ for the child–father duos. Using the SNP profiles from the randomly selected trios and 700 previously typed individuals, a total of 83,096 comparisons between mother, child and an unrelated man were performed. On average, 9–10 mismatches per comparison were detected. Four mismatches were genetic inconsistencies and 5–6 mismatches were opposite homozygosities. In only two of the 83,096 comparisons did an unrelated man match perfectly to a mother–child duo, and in both cases the PI of the true father was much higher than the PI of the unrelated man. The trios were also typed for 15 short tandem repeats (STRs) and seven variable number of tandem repeats (VNTRs). The typical PIs based on 15 STRs or seven VNTRs were 5–50 times higher than the typical PIs based on 52 SNPs. Six mutations in tandem repeats were detected among the randomly selected trios. In contrast, there was not found any mutations in the SNP loci. The results showed that the 52 SNP-plex assay is a very useful alternative to currently used methods in relationship testing. The usefulness of SNP markers with low mutation rates in paternity and immigration casework is discussed.     

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth.

Key points

• Bones and teeth often represent the only sources of DNA for identifying human remains.
• The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors.
• This study focuses on modifications to the previously available Qiagen-based protocols.
• The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols.
• The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

     

Effects of storage time on DNA profiling success from archived latent fingerprint samples using an optimised workflow

Due to recent improvements in forensic DNA testing kit sensitivity,there has been an increased demand in the criminal justice community to revisit past convictions or cold cases.Some of these cases have little biological evidence other than touch DNA in the form of archived latent fingerprint lift cards.In this study,a previously developed optimised workflow for this sample type was tested on aged fingerprints to determine if improved short tandem repeat(STR)profiles could be obtained.Two-year-old samples processed with the optimised workflow produced an average of approximately five more STR alleles per profile over the traditional method.The optimised workflow also produced detectable alleles in samples aged out to 28 years.Of the methods tested,the optimised workflow resulted in the most informative profiles from evidence samples more representative of the forensic need.This workflow is recommended for use with archived latent fingerprint samples,regardless of the archival time.     

Mutation rates at 14 STR loci in the population from Pernambuco, Northeast Brazil

Definition about mutation rates of short tandem repeats (STRs) loci used in forensic analysis are useful for the correct interpretation of resulting genetic profiles and the definition of criterions for exclusion in paternity testing. Germline mutation of 14 STR loci was studied for 54,105 parent–child allelic transfers from 2575 paternity testing cases carried out during 2000–2007 from the Pernambuco State, Northeast Brazil. The parenthood in each of these cases was highly validated (probability > 99.99%). We identified 43 mutations at 12 loci. Locus-specific mutation rate estimates varied between 2 × 10⁻⁴ and 2 × 10⁻³, and the overall mutation rate estimate was 8 × 10⁻⁴. Mutation events in the male germline were more frequent than in the female germline. The majority of the mutations could be explained by losses or gains of one repeat unit and there was no evidence for selection between insertion or deletion changes. Our data were compared with those of Portuguese and North-American populations for CSF1PO, D18S51, D21S11, D7S820, TH01, TPOX and demonstrated, despite the great difference in the size of the sample, that mutation rates of STR loci in a mixed population do not differ from that encountered in different populations.     

Segmental hair analysis for flunitrazepam and 7-aminoflunitrazepam in users: a comparison to existing literature

The availability of more quantitative data on flunitrazepam (FLU) and 7-aminoflunitrazepam (7AF) would aid in obtaining a better understanding of the interpretation of FLU concentrations in human hair. The purpose of this study was to provide concentrations of FLU and 7AF in hair segments of 22 FLU users. Quantitative data regarding hair concentrations of FLU and 7AF from various types of cases were also reviewed to give a comprehensive overview of the comparability of different studies. Three to six 1 cm segments of scalp hair from 22 FLU users were analyzed by a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. FLU and its metabolite were confirmed in the hair segments from all cases. Concentrations of FLU and 7AF in the segments ranged from 0.01–0.16 ng/mg (median of 0.03) and 0.01–0.34 ng/mg (median of 0.09), respectively. Most cases had FLU and 7AF distributions along the hair segments that were suggestive of repeated drug use. A summary of the published concentrations gives valuable data and can assist forensic investigators in their estimations of drug use history and patterns.

Key points

• A method using LC–MS/MS to quantify flunitrazepam and its metabolite was described.
• Segmental analysis of flunitrazepam and its metabolite in human hair was reported.
• A comprehensive overview of quantitative data was given.

     

Pros and cons in the use of SNPs in forensic kinship investigation: a comparative analysis with STRs

Recent advances in single nucleotide polymorphisms (SNPs) research have raised the possibility that these markers could replace the forensically established short tandem repeats (STRs). In this work, we compare STRs and SNPs applicability for kinship investigation in terms of expected informative content and probability of occurrence of "difficult cases" (when isolated Mendelian incompatibilities between alleged father and child are found). Since SNPs have a much lower mutation rate than STRs, these difficulties were expected to occur less frequently if SNPs were used instead of STRs. The purpose of this paper is to make some simulations allowing the estimation of how often such difficult cases are expected to occur using both types of markers and how serious can be their impact in routine work. Our results demonstrate that a battery based exclusively on SNPs matching the informative power of current STR kits would be prone, if applied to routine paternity investigation, to the occurrence of cases where the statistical evidence would be inconclusive. We infer that the introduction of a SNP based strategy, as a substitute to the now classical STR approach poses statistical problems that must be carefully evaluated.     

Resolving relationship tests that show ambiguous STR results using autosomal SNPs as supplementary markers

When using a standard battery of STRs for relationship testing a small proportion of analyses can give ambiguous results – where the claimed relationship cannot be confirmed by a high enough paternity index or excluded with fully incompatible genotypes. The majority of such cases arise from unknowingly testing a brother of the true father and observing only a small number of exclusions that can each be interpreted as one- or two-step mutations. Although adding extra STRs might resolve a proportion of cases, there are few properly validated extra STRs available, while the commonly added hypervariable SE33 locus is four times more mutable than average, increasing the risk of ambiguous results. We have found SNPs in large multiplexes are much more informative for both low initial probabilities or ambiguous exclusions and at the same time provide a more reliable genotyping approach for the highly degraded DNA encountered in many identification cases. Eight relationship cases are outlined where the addition of SNP data resolved analyses that had remained ambiguous even with extended STR typing. In addition we have made simulations to ascertain the frequency of failing to obtain exclusions or conclusive probabilities of paternity with different marker sets when a brother of the true father is tested. Results indicate that SNPs are statistically more efficient than STRs in resolving cases that distinguish first-degree relatives in deficient pedigrees.     

Validation and casework application of a Y chromosome specific STR multiplex

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor’s alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.     

Evaluation of the PowerPlex Fusion System in a sample from East Timor

East Timor is a country located in Southeast Asia. In this study, we determined allele frequencies and forensic parameters for 24 STR autosomal loci included in the PowerPlex^® Fusion System. Autosomal STR data was collected from saliva samples of 100 individuals from East Timor. The amplification of the 24 autosomal STRs was performed using PowerPlex^® Fusion System (Promega Corporation) and the amplified products were analysed on 3500 Genetic Analyser using GeneMapper^® ID-X 1.2 Software (Applied Biosystems). All the analysed loci meet Hardy–Weinberg equilibrium after Bonferroni correction. In our samples, we found “off-ladder” alleles (D2S441, Penta E and FGA locus) confirmed by reamplification, amplification with others kits and sequenced when justified.