Forensic Genetic Casework Research Articles

An optimal skeletal element for DNA testing: Evaluation of DNA quantity and quality from various bone types in routine forensic practice

For human identification, the quality and quantity of DNA must be sufficient for amplification and analysis. When DNA extraction from bone tissues and teeth is required, the optimal skeletal elements should be selected as samples for DNA extraction because DNA yield differs among elements. Recently, some studies have reported that a high quantity of high-quality DNA can be extracted from the small cancellous bones of the hands and feet. In this study, we evaluated the effectiveness of small cancellous bones in the human identification of skeletal remains in routine forensic genetic casework. Cancellous bones [phalanges, (meta)carpal bones, and (meta)tarsal bones)] and the cortical bones (femur and petrous bones) and teeth, which have generally been recommended as samples, were collected from the same individuals that needed identifying using DNA analysis in our laboratory. The quantity of DNA from small cancellous bones tended to be higher than that from cortical bones, and the quality from the former was as high as that from the latter. This study showed that in routine forensic casework, the small cancellous bones of the hands and feet should be actively selected as samples for DNA testing.

Spitting in the wind?-The challenges of RNA sequencing for biomarker discovery from saliva.

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA ("RNA sequencing") has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

Validation of the Microreader™ 36A ID System for forensic genetic casework and database construction

ABSTRACT Autosomal short tandem repeats (STRs) are crucial in identifying humans. Herein, a developmental validation was performed for a newly generated autosomal STR kit (Microreader™ 36A ID System). The sensitivity of this kit was fine when the input DNA dropped down to 0.02 ng/uL. Mixture analysis showed that when male:female = 200 pg: 5400 pg, full profiles of both individuals could be obtained. When inhibited substances existed, all alleles were acquired up to 2000 μM haematin, 1000 ng/μL humic acid or 250 ng/μL tannic acid. The kit exhibited a high precision level with no more than 0.29 nt standard deviation of allele size, with the accuracy (the difference of allele size between the true allele and ladder allele) no more than 0.4 nt. The total discrimination power of this system in the Chinese Han and Hui population was larger than 1–0.1 × 10−30. For the full-sibling test, the identical-by-state (IBS) score varied from 37 to 46, while it was from 21 to 28 when using the AGCU Express marker 22 STR Kit. This kit can increase the autosomal STR information of a single PCR reaction and is promising in the casework of forensic genetic as well as database construction.

Ancestry resolution of South Brazilians by forensic 165 ancestry-informative SNPs panel.

Forensic DNA phenotyping (FDP) includes biogeographic ancestry (BGA) inference and externally visible characteristics (EVCs) prediction directly from an evidential DNA sample as alternatives to provide valuable intelligence when conventional DNA profiling fails to achieve identification. In this context, the application of Massively Parallel Sequencing (MPS) methodologies, which enables simultaneous typing of multiple samples and hundreds of forensic markers, has been gradually implemented in forensic genetic casework. The Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) is a forensic multiplex assay consisting of 165 autosomal SNPs designed to provide biogeographic ancestry information. In this work, a sample of 250 individuals from Rio Grande do Sul (RS) State, southern Brazil, apportioned into four main population groups (African-, European-, Amerindian-, and Admixed-derived Gauchos), was evaluated with this panel, to assess the feasibility of this approach in a highly heterogeneous population. Forensic descriptive parameters estimated for each population group revealed that this panel has enough polymorphic and informative SNPs to be used as a supplementary instrument in forensic individual identification and kinship testing regardless of ethnicity. No statistically significant deviation from Hardy-Weinberg equilibrium was observed after Bonferroni correction. However, seven loci pairs displayed linkage disequilibrium in pairwise LD testing (p<3.70× 10-6). Interpopulation comparisons by FST analysis, MDS plot, and STRUCTURE analysis among the four RS population groups apart and along with 89 reference worldwide populations demonstrated that Admixed- and African-derived Gauchos present the highest levels of admixture and population stratification, whereas European- and Amerindian-derived exhibit a more homogeneous genetic conformation.

Evaluation of a custom QIAseq targeted DNA panel with 164 ancestry informative markers sequenced with the Illumina MiSeq

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.

Evaluation of Promega PowerSeq™ Auto/Y systems prototype on an admixed sample of Rio de Janeiro, Brazil: Population data, sensitivity, stutter and mixture studies

Forensic DNA typing typically relies on the length-based (LB) separation of PCR products containing short tandem repeat loci (STRs). Massively parallel sequencing (MPS) elucidates an additional level of STR motif and flanking region variation. Also, MPS enables simultaneous analysis of different marker-types - autosomal STRs, SNPs for lineage and identification purposes, reducing both the amount of sample used and the turn-around-time of analysis. Therefore, MPS methodologies are being considered as an additional tool in forensic genetic casework. The PowerSeq™ Auto/Y System (Promega Corp), a multiplex forensic kit for MPS, enables analysis of the 22 autosomal STR markers (plus Amelogenin) from the PowerPlex® Fusion 6C kit and 23 Y-STR markers from the PowerPlex® Y23 kit. Population data were generated from 140 individuals from an admixed sample from Rio de Janeiro, Brazil. All samples were processed according to the manufacturers' recommended protocols. Raw data (FastQ) were generated for each indexed sample and analyzed using STRait Razor v2s and PowerSeqv2.config file. The subsequent population data showed the largest increase in expected heterozygosity (23%), from LB to sequence-based (SB) analyses at the D5S818 locus. Unreported allele was found at the D21S11 locus. The random match probability across all loci decreased from 5.9 × 10-28 to 7.6 × 10-33. Sensitivity studies using 1, 0.25, 0.062 and 0.016 ng of DNA input were analyzed in triplicate. Full Y-STR profiles were detected in all samples, and no autosomal allele drop-out was observed with 62 pg of input DNA. For mixture studies, 1 ng of genomic DNA from a male and female sample at 1:1, 1:4, 1:9, 1:19 and 1:49 proportions were analyzed in triplicate. Clearly resolvable alleles (i.e., no stacking or shared alleles) were obtained at a 1:19 male to female contributor ratio. The minus one stutter (−1) increased with the longest uninterrupted stretch (LUS) allele size reads and according to simple or compound/complex repeats. The haplotype-specific stutter rates add more information for mixed samples interpretation. These data support the use of the PowerSeqTM Auto/Y systems prototype kit (22 autosomal STR loci, 23 Y-STR loci and Amelogenin) for forensic genetics applications.

Overview of Forensic DNA Profiling and Database

Deoxyribonucleic acid (DNA) profiling, has had a tremendous impact on forensic genetics. Before DNA profiling, all forensic genetic casework (e.g., Paternity testing, criminal casework, individual identification) was performed using classical serological genetic markers. Blood groups, human leukocyte antigen (HLA), and polymorphic protein and enzymes were used for solving forensic genetic casework using immunological and electrophoretic methodologies. These genetic markers were nevertheless limited when it was necessary to analyze minimal or degraded material, which is commonly involved in forensic cases. An STR is a region of human DNA containing an array of tandem repeats. Arrays range from only a 10 to about a hundred repeated units. This essay confers the basic concepts of operating of DNA in the criminal investigation. This review primarily summarizes the major tandem repeat markers used in forensic DNA profiling, that assist criminal’s conviction, exonerate the inferring individuals, and recognize victims of violence, catastrophes, and armed conflict.

Developmental validation of the Yfiler Platinum PCR Amplification Kit for forensic genetic caseworks and databases.

Y chromosome kits are successfully applied in cases where human biological material exists. With the development of genotyping ability, more Y chromosomal markers are needed for finer identification of male individuals and lineages. In this study, a developmental validation of a newly emerged Y chromosome kit that combines two different kinds of markers: 38 Y-STRs and 3 Y-indels are conducted. The results show that this kit has high sensitivity when there is a small amount of DNA (125 pg), more than one male (minor:major = 1:7), or a mixture of males and females (male:female = 125pg:1875pg), inhibited substances (800 μM hematin and more than 1600ng/μL humic acid). The kit exhibits high precision level with a standard deviation of allele size no more than 0.14nt. Locus DYS481 shows the largest stutter rate, with three stutters per true allele. Population samples are well identified (MP of 0.001106), and mutations can be observed in father-son pairs (46 mutations in 70 pairs, 10 in locus DYS627). Out of all the population samples, 13.2% belong to haplogroup M117-O2a2b1a1, with their ethnic group being Han Chinese. The results show that this kit can improve the performance of identifying male individuals, obtaining more unique haplotypes (increasing from 894 to 918 of 1000 male samples) and higher discrimination capacity (increasing from 0.942 to 0.955) in this study compared to previous widely used Yfiler Plus kit. Besides, it gives information about their paternal lineages in forensic genetic casework and genealogical database construction.

Typing of two Middle Eastern populations with the Precision ID Ancestry Panel

Ancestry informative markers have a great potential for ancestry inference in forensic genetic casework. However, applying ancestry inference in casework requires sufficient allele frequency data on as many populations as possible to allow the determination of biogeographical origin. Therefore, Turkish and Iranian individuals were SNP typed with Massively Parallel Sequencing with the Precision ID Ancestry Panel (Thermo Fisher Scientific) to assess whether it was possible to differentiate geographically proximate populations in the Middle East using this kit. Analyses showed that it were not possible to separate the two populations and neither was it possible to separate them from European and South-Central Asian populations. The two Middle Eastern populations were highly admixed with components from both Europe and South-Central Asia. Thus, it is important to identify genetic markers that are characteristic of the various Middle Eastern populations.

Comparison of manual and automated AmpliSeq™ workflows in the typing of a Somali population with the Precision ID Identity Panel

The Precision ID Identity Panel was used to type 109 Somali individuals in order to obtain allele frequencies for the Somali population. These frequencies were used to establish a Somali HID-SNP database, which will be used for the biostatistic calculations in family and immigration cases. Genotypes obtained with the Precision ID Identity Panel were found to be almost in complete concordance with genotypes obtained with the SNPforID PCR-SBE-CE assay. In seven SNP loci, silent alleles were identified, of which most were previously described in the literature.The project also set out to compare different AmpliSeq™ workflows to investigate the possibility of using automated library building in forensic genetic case work. In order to do so, the SNP typing of the Somalis was performed using three different workflows: 1) manual library building and sequencing on the Ion PGM™, 2) automated library building using the Biomek®3000 and sequencing on the Ion PGM™, and 3) automated library building using the Ion Chef™ and sequencing on the Ion S5™. AmpliSeq™ workflows were compared based on coverage, locus balance, noise, and heterozygote balance. Overall, the Ion Chef™/Ion S5™ workflow was found to give the best results and required least hands-on time in the laboratory. However, the Ion Chef™/Ion S5™ workflow was also the most expensive. The number of libraries that may be constructed in one Ion Chef™ library building run was limited to eight, which is too little for high throughput workflows. The Biomek®3000/Ion PGM™ workflow was found to perform similarly to the manual/Ion PGM™ workflow. This argues for the use of automated library building in forensic genetic case work. Automated library building decreases the workload of the laboratory staff, decreases the risk of pipetting errors, and simplifies the daily workflow in forensic genetic laboratories.

Evaluation of the Precision ID Ancestry Panel for crime case work: A SNP typing assay developed for typing of 165 ancestral informative markers

The application of massive parallel sequencing (MPS) methodologies in forensic genetics is promising and it is gradually being implemented in forensic genetic case work. One of the major advantages of these technologies is that several traditional electrophoresis assays can be combined into one single MPS assay. This reduces both the amount of sample used and the time of the investigations.This study assessed the utility of the Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) in forensic genetics. This assay was developed for the Ion Torrent PGM™ System and genotypes 165 ancestry informative SNPs. The performance of the assay and the accompanying software solution for ancestry inference was assessed by typing 142 Danes and 98 Somalis. Locus balance, heterozygote balance, and noise levels were calculated and future analysis criteria for crime case work were estimated. Overall, the Precision ID Ancestry Panel performed well, and only minor changes to the recommended protocol were implemented. Three out of the 165 loci (rs459920, rs7251928, and rs7722456) had consistently poor performance, mainly due to misalignment of homopolymeric stretches. We suggest that these loci should be excluded from the analyses.The different statistical methods for reporting ancestry in forensic genetic case work are discussed.

Testing of the Illumina® ForenSeq™ kit

The Illumina® ForenSeq™ kit amplifies in one multiplex PCR reaction 59 STRs and 172 SNPs relevant to forensic genetic case work. Typed markers include markers for identification, ancestry identification (AIMs) and pigmentary traits. The products were sequenced on the MiSeq FGx Forensic Genomics System and analysed with the ForenSeq™ Universal Analysis Software. Using this approach, we sequenced 10 Danish, 10 Italian, and 10 individuals from other populations, mostly non-europeans. The eye and hair colours as well as the bio-geographical ancestry of the individuals were known.Blue and brown eye colours were predicted accurately by the ForenSeq™ assay, whereas intermediate eye colours were not. The success of the hair colour prediction tool seemed population-dependent. The predictions matched the hair colours reported by the individuals themselves in twice as many Danes as Italians. The geographic ancestries of 22 out of 23 individuals of European or East Asian ancestry were correctly predicted. However, the ForenSeq™ Universal Analysis Software was unable to differentiate between individuals from Middle East/South Asia, South America and North Africa.We found no difference in STR typing results obtained with the ForenSeq™ kit and capillary electrophoresis.

Next generation sequencing and its applications in forensic genetics

It has been almost a decade since the first next generation sequencing (NGS) technologies emerged and quickly changed the way genetic research is conducted. Today, full genomes are mapped and published almost weekly and with ever increasing speed and decreasing costs. NGS methods and platforms have matured during the last 10 years, and the quality of the sequences has reached a level where NGS is used in clinical diagnostics of humans. Forensic genetic laboratories have also explored NGS technologies and especially in the last year, there has been a small explosion in the number of scientific articles and presentations at conferences with forensic aspects of NGS. These contributions have demonstrated that NGS offers new possibilities for forensic genetic case work. More information may be obtained from unique samples in a single experiment by analyzing combinations of markers (STRs, SNPs, insertion/deletions, mRNA) that cannot be analyzed simultaneously with the standard PCR-CE methods used today. The true variation in core forensic STR loci has been uncovered, and previously unknown STR alleles have been discovered. The detailed sequence information may aid mixture interpretation and will increase the statistical weight of the evidence. In this review, we will give an introduction to NGS and single-molecule sequencing, and we will discuss the possible applications of NGS in forensic genetics.

The effect of gender on eye colour variation in European populations and an evaluation of the IrisPlex prediction model

In two recent studies of Spanish individuals, gender was suggested as a factor that contributes to human eye colour variation. However, gender did not improve the predictive accuracy on blue, intermediate and brown eye colours when gender was included in the IrisPlex model. In this study, we investigate the role of gender as a factor that contributes to eye colour variation and suggest that the gender effect on eye colour is population specific. A total of 230 Italian individuals were typed for the six IrisPlex SNPs (rs12913832, rs1800407, rs12896399, rs1393350, rs16891982 and rs12203592). A quantitative eye colour score (Pixel Index of the Eye: PIE-score) was calculated based on digital eye images using the custom made DIAT software. The results were compared with those of Danish and Swedish population samples. As expected, we found HERC2 rs12913832 as the main predictor of human eye colour independently of ancestry. Furthermore, we found gender to be significantly associated with quantitative eye colour measurements in the Italian population sample. We found that the association was statistically significant only among Italian individuals typed as heterozygote GA for HERC2 rs12913832. Interestingly, we did not observe the same association in the Danish and Swedish population. This indicated that the gender effect on eye colour is population specific. We estimated the effect of gender on quantitative eye colour in the Italian population sample to be 4.9%. Among gender and the IrisPlex SNPs, gender ranked as the second most important predictor of human eye colour variation in Italians after HERC2 rs12913832. We, furthermore, tested the five lower ranked IrisPlex predictors, and evaluated all possible 3(6) (729) genotype combinations of the IrisPlex assay and their corresponding predictive values using the IrisPlex prediction model [4]. The results suggested that maximum three (rs12913832, rs1800407, rs16891982) of the six IrisPlex SNPs are useful in practical forensic genetic casework.

On the exact distribution of the numbers of alleles in DNA mixtures

When more than one individual contributes biological material to a forensic stain, the resulting DNA type is termed a DNA mixture. DNA mixtures occur frequently in forensic genetic casework, and in recent years, much research has been devoted to this subject. This paper presents a derivation of the exact distribution of the number of alleles for any number of profiles and investigated loci. The per locus number of observed alleles is of interest as it indicates the plausible range on the number of contributors. Hence, by specifying a prior distribution on the number of contributors, the locus distribution may be used to assess the number of contributors. Furthermore, the total number of alleles across all loci is used by some forensic geneticists to estimate the probability that an allele has failed to be detected (allelic drop-out).

A 21 marker insertion deletion polymorphism panel to study biogeographic ancestry

Insertion/deletion polymorphisms have recently received increased interest in the forensic genetics community. This class of markers combines the advantageous genetic properties of single nucleotide polymorphisms (i.e., low mutation rate, genetic stability, and short amplicon size) with the technical advantage of short tandem repeat markers (simple detection by fluorescence-labelled PCR and capillary electrophoresis). For a large number of indel markers significant differences in allele frequencies between the major populations have been reported, making this class of markers suitable for the analysis of biogeographic ancestry. We have developed a multiplex PCR assay designed to establish the biogeographic ancestry of forensic DNA samples based on insertion/deletion polymorphisms. A panel of 21 short indels with allele frequency differences between three major population groups (European, African and Asian) was selected to be incorporated into a single-tube multiplex PCR assay. The assay is highly sensitive, requiring less than 0.5 ng of genomic DNA for successful typing. Due to the short fragment lengths below 200 bp, the assay is ideally suited for the typing of challenging forensic genetic case work samples. A population genetic study has been performed proving the performance of the assay in inferring the ancestral population of individuals. The chosen 21 markers are sufficient to distinguish between three major global population groups. Furthermore, the assay design leaves room for an extension in order to cover additional population groups.

Performance of the PowerPlexY23® kit on trace samples in forensic genetic casework

Y-chromosomal short tandem repeats (Y-STRs) can be analysed with the AmpFlSTR®Yfiler® kit (Yfiler, Life Technologies (LT)) from trace samples in forensic case-work. However, the results are not reliable when the ratio of female:male DNA is high. The aim of this study was to investigate the performance of the PowerPlex®Y23 kit (PPY23, Promega) on trace samples from crime scenes. A total of 100–200pg male DNA was analysed with the PPY23 kit from 45 trace samples. Amplification of Y-STRs with the PPY23 kit was successful for 34 (76%) of the trace samples. Reproducible artefact peaks in the reading area (80–450 base pairs) were found in up to 29% of the trace samples typed with the PPY23 kit. Significantly higher female:male DNA ratios were observed in profiles with artefact peaks compared to those of profiles with no artefact peak. It was concluded that the quality of Y-STR analyses of trace samples would not be improved by replacing the Yfiler kit with the PPY23 kit in our laboratory.

On the exact distribution of the numbers of alleles in DNA mixtures

When more than one individual contributes biological material to a forensic stain, the resulting DNA type is termed a DNA mixture. DNA mixtures occur frequently in forensic genetic casework, and in recent years much research has been devoted to this subject.This paper presents the exact distribution of the number of alleles for any number of profiles and investigated loci. The per locus number of observed alleles is of interest as it indicates the plausible range of the number of contributors. Furthermore, the total number of alleles across all loci are used by some forensic geneticists to estimate the allelic drop-out probability.

Body fluid identification of blood, saliva and semen using second generation sequencing of micro-RNA

We report a new second generation sequencing method for identification micro-RNA (miRNA) that can be used to identify body fluids and tissues. Principal component analysis of 10 miRNAs with high expression in 16 samples of blood, saliva and semen showed clear differences in the expression of miRNA in the investigated tissues. We expect that the method can also be used for identification of other miRNAs that can be used for identifying other body fluids and tissues. We also expect that the method can be used for identification of body fluids and tissues in practical forensic genetic case work.

Genetic data from 15 STR loci for forensic individual identification and parentage analyses in UK domestic dogs (Canis lupus familiaris)

Eighteen STR loci and one sex determination locus present in the Finnzymes Canine 2.1 STR Multiplex Reagent Kit were screened in the UK dog population providing allele frequencies and population genetic parameters necessary for the application of STRs to forensic genetic casework. A total of 375 dogs were genotyped, including representative samples from each of twelve breeds used to evaluate Hardy–Weinberg equilibrium and calculate inter-population pairwise FST values. Three loci were excluded from calculations of average random match probability due to deviations from Hardy–Weinberg Equilibrium or ambiguous genotyping. Random match probability based on fifteen STR loci and one sex locus was subsequently estimated to be 2.8×10−17 for unrelated individuals across breeds.