Search

Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards with buccal swabs and compared the results with those obtained with DNA extracted using the EZ1 DNA Investigator Kit. Concordant profiles were obtained for all samples. Our protocol includes simple punch, wash, and PCR steps, reducing cost and hands-on time in the laboratory. Furthermore, it facilitates automation of DNA sequencing.

DNA-based prediction of hair morphology, defined as straight, curly or wavy hair, could contribute to an improved description of an unknown offender and allow more accurate forensic reconstructions of physical appearance in the field of forensic DNA phenotyping. Differences in scalp hair morphology are significant at the worldwide scale and within Europe. The only genome-wide association study made to date revealed the Trichohyalin gene (TCHH) to be significantly associated with hair morphology in Europeans and reported weaker associations for WNT10A and FRAS1 genes. We conducted a study that centered on six SNPs located in these three genes with a sample of 528 individuals from Poland. The predictive capacity of the candidate DNA variants was evaluated using logistic regression; classification and regression trees; and neural networks, by applying a 10-fold cross validation procedure. Additionally, an independent test set of 142 males from six European populations was used to verify performance of the developed prediction models. Our study confirmed association of rs11803731 (TCHH), rs7349332 (WNT10A) and rs1268789 (FRAS1) SNPs with hair morphology. The combined genotype risk score for straight hair had an odds ratio of 2.7 and these predictors explained ∼ 8.2% of the total variance. The selected three SNPs were found to predict straight hair with a high sensitivity but low specificity when a 10-fold cross validation procedure was applied and the best results were obtained using the neural networks approach (AUC=0.688, sensitivity=91.2%, specificity=23.0%). Application of the neural networks model with 65% probability threshold on an additional test set gave high sensitivity (81.4%) and improved specificity (50.0%) with a total of 78.7% correct calls, but a high non-classification rate (66.9%). The combined TTGGGG SNP genotype for rs11803731, rs7349332, rs1268789 (European frequency=4.5%) of all six straight hair-associated alleles was identified as the best predictor, giving >80% probability of straight hair. Finally, association testing of 44 SNPs previously identified to be associated with male pattern baldness revealed a suggestive association with hair morphology for rs4679955 on 3q25.1. The study results reported provide the starting point for the development of a predictive test for hair morphology in Europeans. More studies are now needed to discover additional determinants of hair morphology to improve the predictive accuracy of this trait in forensic analysis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

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., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Six case work examples are presented, where the individuals were typed for 15 autosomal short tandem repeats (STRs) and 49 autosomal single nucleotide polymorphisms (SNPs). The 15 STRs were typed with the AmpFlSTR Identifiler PCR Amplification Kit and the 49 SNPs were typed with the SNPforID multiplex assay. The six cases included two duos, two trios and two cases, where the alleged father was not available for testing and one or two of his close relatives were tested instead. The SNP investigation was more informative than the STR investigation in all six cases. In two cases, the alleged father would have been falsely included based on the STR results, while the SNP results showed that the alleged father was not the true parent. These case work examples underline the importance of performing supplementary investigations in selected cases and demonstrate the usefulness of the SNPforID multiplex assay., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Introduction of new methods requires meticulous evaluation before they can be applied to forensic genetic case work. Here, a custom QIAseq Targeted DNA panel with 164 ancestry informative markers was assessed using the MiSeq sequencing platform. Concordance, sensitivity, and the capability for analysis of mixtures were tested. The assay gave reproducible and nearly concordant results with an input of 10 and 2 ng DNA. Lower DNA input led to an increase in both locus and allele drop-outs, and a higher variation in heterozygote balance. Locus or allele drop-outs in the samples with less than 2 ng DNA input were not necessarily associated with the overall performance of a locus. Thus, the QIAseq assay will be difficult to implement in a forensic genetic setting where the sample material is often scarce and of poor quality. With equal or near equal mixture ratios, the mixture DNA profiles were easily identified by an increased number of imbalanced heterozygotes. For more skewed mixture ratios, the mixture DNA profiles were identified by an increased noise level. Lastly, individuals from Great Britain and the Middle East were investigated. The Middle Eastern individuals showed a greater affinity with South European populations compared to North European populations. [ABSTRACT FROM AUTHOR]

Abstract: Fundamental to most forensic analyses is the availability of genomic DNA of adequate quality and quantity. To perform a multitude of genetic analyses and assays requires a sufficiently large amount of template. However, DNA yield from forensic samples is frequently limiting the extent of genetic typing. A possible solution to overcome this “bottleneck” of forensic and paleoarcheological DNA analyses could be the amplification of the entire genomic DNA prior to locus specific PCR analysis. Whole Genome Amplification appears to be a promising tool to obtain sufficient DNA amounts from forensic samples of limited quantity. [Copyright &y& Elsevier]

Abstract: We performed multiple displacement amplification (MDA) of 50 blood and 50 saliva samples placed on FTA cards. A 1.2-mm disk was punched out of the FTA cards. The disk was washed, dried and used as target for the GenomiPhi DNA amplification kit. On average, the Phi29 polymerase produced 2 μg whole genome amplified DNA (wgaDNA) with DNA fragment sizes ranging from a few hundred bp to 12 kbp. A total of 1–2 ng wgaDNA was typed using the AmpFlSTR® SGM Plus™ amplification kit and the resulting STR profiles analysed and compared to genotypes obtained from genomic DNA. Only 25 of the 100 wgaDNA samples gave concordant STR profiles. Locus drop-outs, missing alleles, allele imbalances or false alleles were observed in 11, 27, 61 and 10 wgaDNA samples, respectively. The same FTA disk was used successfully in five consecutive rounds of MDA. However, after the 4th and 5th round of MDA, a further increase in the number of allele drop-outs as well as system and allele imbalances were observed. [Copyright &y& Elsevier]

Abstract: An assay was designed for typing 29 of the 52 SNPs in the SNPforID multiplex on the Sequenom® MassARRAY® analyzer 4 system. The dedicated TYPER 4 software was used for automatic allele calling. The sensitivity of the assay was tested using 0.5–40ng pristine template DNA in the PCR reaction. Overall, no difference was observed in the sensitivity, measured as call rate, for the different amounts of template DNA. Typing of paternity case work samples resulted in lower call rates (54%) as compared to the sensitivity test (89%), and more incorrect calls, 0.4% as compared to 0.1%. Finally, the ability of the software to detect 1:1, 1:3 and 1:5 DNA mixtures was investigated. Compared to the sensitivity test, the call rates of the 1:1 and 1:3 mixtures were low (64–74%), whereas the call rates of the 1:5 mixtures were similar (88–90%). However, in comparison with the case work samples, the call rates of the mixtures were not unusual and mixtures may be overlooked. [Copyright &y& Elsevier]

Abstract: The SNPforID multiplex was tested on typical crime case samples. In a mixture study, both controlled experiments with samples mixed in known ratios and a blind study were conducted. All mixtures were identified, including samples mixed in 1:40 ratios. For the study of degraded DNA, 50 crime case samples were selected. Complete SNP profiles were obtained in 80% of the highly degraded samples and at least 41 SNPs were typed in the last 20%. Furthermore, 30% of the highly degraded samples turned out to be mixtures. [Copyright &y& Elsevier]

Abstract: In this study, the GenomiPhi™ DNA Amplification Kit (Amersham Biosciences) was used to investigate the potential of whole genome amplification (WGA) when considering samples originating from more than one donor. DNA was extracted from blood samples, quantified and normalised before being mixed in ratios of 1:1, 1:3, 1:7 and 1:15. Proportions were checked using standard STR analysis before being subjected to WGA. Once amplified using GenomiPhi™, the ratios were again checked. Relative proportions were found to be maintained in the 1:1 and 1:3 ratios following WGA; the observed peak ratios were found to match the expected peak ratios regardless of the starting concentration of DNA. With samples mixed in the ratio of 1:7 and 1:15, and when the concentration of starting material was at the manufacturer''s lower limit, too few minor component peaks were found to allow for statistical analysis. With an initial template exceeding 1 ng/μL there was an increase in problems associated with profile interpretation but the results obtained indicated that mixture proportions could be quantifiably maintained. [Copyright &y& Elsevier]

Abstract: We have implemented a laboratory automation system (LAS) for the preparation of database samples. The LAS encompass registration of samples in a LIMS database, export of a work-list to a puncher, punching of FTA-cards by a BSD600-duet puncher, electronic sample check by means of barcodes, washing of punches by the THEONYX liquid-handler from MWG, PCR amplification and electrophoresis of amplicons. We validated the system using fresh blood and saliva immobilized on FTA-cards as sources of biological material. The use of the LAS led to: [–] Correct STR types with a quality equal to or higher than that of the profiles produced using chelex-extracted DNA. [–] A reduction of approximately 35% in the number of reanalysed samples. [–] A reduced risk of mix-up of samples during the laboratory procedure. [–] Fewer sample transfers. [–] A reduction in the number of PCR cycles from 28 to 24 compared to chelex-extracted DNA from blood. During the validation process, we did not observe mix-up of samples, loss of FTA-card punches, contamination from external sources, or cross-contamination between samples. [Copyright &y& Elsevier]

Abstract: An efficient method that can be used to simultaneously amplify a set of genetic loci across the genome with high reliability can provide a valuable tool for single nucleotide polymorphism (SNP) forensic genotyping. A crucial element is the number of individual biochemical reactions that must be performed. The SNPforID consortium (www.snpforid.org) was established in 2003 with the principal goal of developing a SNP-based system of DNA analysis that would have comparable discrimination power and ease of use to those of existing short tandem repeat (STR) based techniques. Here, we describe a strategy for amplifying 52 genomic DNA fragments, each containing one SNP, in a single tube, and accurately genotyping the PCR product mixture using two single base extension reactions. This multiplex approach reduces the cost of SNP genotyping and requires as little as 0.5 ng of genomic DNA to detect 52 SNPs. We used a multiple injection approach for DNA sequencers that can effectively detect all the SNPs amplified in a single electrophoretic run. We present SNP data for 700 unrelated individuals from 9 populations. [Copyright &y& Elsevier]