Pyrosequencing: Current forensic methodology and future applications-a review.

The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science. Pyrosequencing is a sequencing by synthesis technique, based on chemiluminescent inorganic pyrophosphate detection. This review explains the pyrosequencing workflow and illustrates the step-by-step chemistry, followed by a description of the assay design and factors to keep in mind for an exemplary assay. Existing and potential forensic applications are highlighted using this technology. Current applications include identifying species, identifying bodily fluids, and determining smoking status. We also review progress in potential applications for the future, including research on distinguishing monozygotic twins, detecting alcohol and drug abuse, and other phenotypic characteristics such as diet and body mass index. Overall, the versatility of the pyrosequencing technologies renders it a useful tool in forensic genomics.

Match statistics for sequence-based alleles in profiles from forensic PCR-mps kits.

The first autosomal sequence-based allele (aka SNP-STR haplotype) frequency database for forensic massively parallel sequencing (MPS) has been published, thereby removing one of the remaining barriers to implementing MPS in casework. The database was developed using a specific set of flank trim sites. If different trim sites or different kits with different primers are used for casework, then SNP-STR haplotypes may be detected that do not have frequencies in the database. We describe a procedure to address calculation of match probabilities when casework samples are generated using an MPS kit with different trim sites than those present in the relevant population frequency database. The procedure provides a framework for comparison of any MPS kit or database combination while also accommodating comparison of MPS and CE profiles.

A data-driven, high-throughput methodology to determine tissue-specific differentially methylated regions able to discriminate body fluids.

Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.

Efficacy of home-based visuomotor feedback training in stroke patients with chronic hemispatial neglect.

Hemispatial neglect is a severe cognitive condition frequently observed after a stroke, associated with unawareness of one side of space, disability and poor long-term outcome. Visuomotor feedback training (VFT) is a neglect rehabilitation technique that involves a simple, inexpensive and feasible training of grasping-to-lift rods at the centre. We compared the immediate and long-term effects of VFT vs. a control training when delivered in a home-based setting. Twenty participants were randomly allocated to an intervention (who received VFT) or a control group (n = 10 each). Training was delivered for two sessions by an experimenter and then patients self-administered it for 10 sessions over two weeks. Outcome measures included the Behavioural Inattention Test (BIT), line bisection, Balloons Test, Landmark task, room description task, subjective straight-ahead pointing task and the Stroke Impact Scale. The measures were obtained before, immediately after the training sessions and after four-months post-training. Significantly greater short and long-term improvements were obtained after VFT when compared to control training in line bisection, BIT and spatial bias in cancellation. VFT also produced improvements on activities of daily living. We conclude that VFT is a feasible, effective, home-based rehabilitation method for neglect patients that warrants further investigation with well-designed randomised controlled trials on a large sample of patients.

Forensic discrimination of vaginal epithelia by DNA methylation analysis through pyrosequencing.

The accurate identification of body fluids from crime scenes can aid in the discrimination between criminal and innocent intent. This research aimed to determine if the levels of DNA methylation in the locus PFN3A could be used to discriminate vaginal epithelia from other body fluids. In this work we bisulfite-modified and amplified DNA samples from blood, saliva, semen, and vaginal epithelia using primers for PFN3A. Through pyrosequencing we were able to show that vaginal epithelia present distinct methylation levels when compared to other body fluids. Mixtures of different body fluids present methylation values that correlate with single-source body fluid samples and the primers for PFN3A are specific for primates. This report successfully demonstrated that the analysis of methylation in the PFN3A locus can be used for vaginal epithelia discrimination in forensic samples.

High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

Evaluation of DNA methylation markers and their potential to predict human aging.

We present epigenetic methylation data for two genetic loci, GRIA2, and NPTX2, which were tested for prediction of age from different donors of biofluids. We analyzed 44 saliva samples and 23 blood samples from volunteers with ages ranging from 5 to 72 years. DNA was extracted and bisulfite modified using commercial kits. Specific primers were used for amplification and methylation profiles were determined by pyrosequencing. Methylation data from both markers and their relationship with age were determined using linear regression analysis, which indicates a positive correlation between methylation and age. Older individuals tend to have increased methylation in both markers compared to younger individuals and this trend was more pronounced in the GRIA2 locus when compared to NPTX2. The epigenetic predicted age, calculated using a GRIA2 regression analysis model, was strongly correlated to chronological age (R(2) = 0.801), with an average difference of 6.9 years between estimated and observed ages. When using a NPTX2 regression model, we observed a lower correlation between predicted and chronological age (R(2) = 0.654), with an average difference of 9.2 years. These data indicate these loci can be used as a novel tool for age prediction with potential applications in many areas, including clinical and forensic investigations.

Identification of spermatozoa by tissue-specific differential DNA methylation using bisulfite modification and pyrosequencing.

The focus of this study is to evaluate the application of epigenetic markers as a forensic tool for the determination of semen present in sexual assault cases. A series of genetic loci were screened in order to identify certain epigenetic markers displaying differential methylation that can allow semen to be differentiated from blood, buccal cells, skin epidermis, and vaginal epithelial cells. Of the different loci tested, a panel of six markers, DACT1, USP49, DDX4, Hs_INSL6_03, Hs_ZC3H12D_05, and B_SPTB_03 were identified to contain tissue-specific differential methylation. Samples ranging from 9-21 for each tissue type were collected and subjected to bisulfite modification. The bisulfite modified DNA was amplified by PCR, and analyzed by pyrosequencing to quantitate the level of methylation at each marker. All six markers successfully differentiated semen samples from the other four tissue types analyzed. Sperm DNA was hypomethylated in all but one marker, B_SPTB_03, where this marker showed hypermethylation. Mean methylation percentages for semen samples were statistically significant from mean methylation percentages of the other four tissues studied (p < 0.01). The results of this study demonstrate the applicability of epigenetic markers as a novel tool for determination of spermatozoa and to identify the tissue source of origin of a DNA sample.

High-resolution melt analysis of the minisatellite D1S80: a potential forensic screening tool.

High-resolution melt (HRM) analysis of the VNTR region of the human D1S80 locus, a 16-bp repeat minisatellite from approximately 400 to over 700 bp in length, was investigated. A Qiagen Rotor-Gene Q using the Type-it PCR HRM kit was used to acquire HRM curves for 14 single, and 16 biallelic, dsDNA samples. The HRM analysis was applicable over a range of DNA concentrations; however the characteristics of the melt curve did depend on the forward and reverse primer ratio. Despite the large amplicon size and the similarities of the repeat sequences, it was possible to discriminate different genotypes. Heterozygotes were clearly different from the homozygous variants and even small differences in the repeat sequence could be differentiated. However, the melt analysis requires a high-resolution system with temperature resolution of 0.02°C or better in order to sort out differences in these large amplicons of near identical GC content (in this case 56%). HRM analysis of amplicons with large repeat sequences can be used as a means of comparing DNA fragments. Examination of multiple sequences can be used to differentiate DNA samples and demonstrate the potential of HRM analysis as a rapid and inexpensive prescreening technique in forensic applications.

A study of the potential application of digital PCR in the detection of fecal contamination of strawberries using Bacteroides markers.

Food-borne illnesses can result from contamination of agricultural products. In this study, we examined nanoplate digital PCR (dPCR) to test for fecal contamination of agricultural products. In nanoplate technique, the PCR mastermix is divided into 8.526,000 partitions, providing direct detection of individual DNA molecules, with correction by Poisson distribution. In this project, strawberries were inoculated with fecal material from animals, and the result detected by nanoplate digital PCR. A detection limit of 250 fg/uL was determined. Overall, dPCR offers a quick and sensitive method to detect contaminated produce.

The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing.

The goal of this study is to explore the application of epigenetic markers in the identification of biofluids that are commonly found at the crime scene. A series of genetic loci were examined in order to define epigenetic markers that display differential methylation patterns between blood, saliva, semen, and epithelial tissue. Among the different loci tested, we have identified a panel of markers, C20orf117, ZC3H12D, BCAS4, and FGF7, that can be used in the determination of these four tissue types. Since methylation modifications occur at cytosine bases that are immediately followed by guanine bases (CpG sites), methylation levels were measured at CpG sites spanning each marker. Up to 11 samples of each tissue type were collected and subjected to bisulfite modification to convert unmethylated CpG-associated cytosine bases to thymine bases. The bisulfite modified DNA was then amplified via nested PCR using a primer set of which one primer was biotin labeled. Biotinylated PCR products were in turn analyzed and the methylation level at each CpG site was quantitated by pyrosequencing. The percent methylation values at each CpG site were determined and averaged for each tissue type. The results indicated significant methylation differences between the tissue types. The methylation patterns at the ZC3H12D and FGF7 loci differentiated sperm from blood, saliva, and epithelial cells. The C20orf117 locus differentiated blood from sperm, saliva, and epithelial cells and saliva was differentiated from blood, sperm, and epithelial cells at a fourth locus, BCAS4. The results of this study demonstrate the applicability of epigenetic markers as a novel tool for the determination of biofluids using bisulfite modification and pyrosequencing.

The role of right temporal lobe structures in off-line action: evidence from lesion-behavior mapping in stroke patients.

Recent evidence suggests the possibility that not all action modes depend on dorsal visual stream processing but that off-line nontarget-directed actions, such as antipointing, require additional and even distinct neural networks when compared with target-directed online actions. Here, we explored this potential dissociation in a group of 11 patients with left visual neglect, a syndrome characterized by a loss of awareness of the contralesional side of space. Ten healthy participants and 10 right hemisphere-damaged patients without neglect served as controls. Participants had to point either directly toward targets presented on their left or right (i.e., propointing) or to the mirror position in the opposite hemispace (i.e., antipointing). Compared with both control groups, neglect patients showed reduced accuracy when antipointing but not propointing. Lesion-behavior mapping revealed that the areas critically associated with these deficits were located in the middle and superior temporal and parahippocampal gyri. We argue that neglect patients present specific deficits only when the visuomotor task taps into more perceptual representations thought to rely on ventral visual stream processing and that our results indicate that right temporal brain regions are implicated in these off-line actions.

Mutation at the human D1S80 minisatellite locus.

Little is known about the general biology of minisatellites. The purpose of this study is to examine repeat mutations from the D1S80 minisatellite locus by sequence analysis to elucidate the mutational process at this locus. This is a highly polymorphic minisatellite locus, located in the subtelomeric region of chromosome 1. We have analyzed 90,000 human germline transmission events and found seven (7) mutations at this locus. The D1S80 alleles of the parentage trio, the child, mother, and the alleged father were sequenced and the origin of the mutation was determined. Using American Association of Blood Banks (AABB) guidelines, we found a male mutation rate of 1.04 × 10(-4) and a female mutation rate of 5.18 × 10(-5) with an overall mutation rate of approximately 7.77 × 10(-5). Also, in this study, we found that the identified mutations are in close proximity to the center of the repeat array rather than at the ends of the repeat array. Several studies have examined the mutational mechanisms of the minisatellites according to infinite allele model (IAM) and the one-step stepwise mutation model (SMM). In this study, we found that this locus fits into the one-step mutation model (SMM) mechanism in six out of seven instances similar to STR loci.

Developmental validation of SpeID: A pyrosequencing-based assay for species identification.

In crime scenes, biological exhibits are often human in origin, yet biological stains from other fauna may also be present at a crime scene, creating confusion during an investigation. Furthermore, identifying the source of a biological sample can be critical during an investigation. To identify the presence of biological material from non-human sources, it is common to use genetic markers within mitochondrial DNA such as cytochrome b, 16S rRNA, and 12S rRNA genes. This process usually requires DNA sequencing, a process that is neither quick nor easy. In general, a faster, more standardized method for species identification from tissue and body fluids is desirable.For this reason, we have developed a vertebrate specific real-time quantitation method that is followed by an automated pyrosequencing-based procedure that sequences a short fragment within the 12S rRNA gene. Using no more than 35 bases, the assay can distinguish between 32 different species commonly found in and around a household with a turnaround time of 6 h from extraction to sequencing. -Using this procedure, up to 48 samples can be run at a time without the need for expensive reagents or bioinformatic skills.

The genital microbiome and its potential for detecting sexual assault.

Since its inception, the Human Microbiome Project (HMP) has provided key discoveries that can be applied to forensics, in addition to those of obvious medical value. Whether for postmortem interval estimation, geolocation, or human identification, there are many applications of the microbiome as an investigative lead for forensic casework. The human skin microbiome has shown great potential for use in studies of transfer and human identification, however there has been little focus on the genital microbiome, in particular penile skin which differs from other body sites. Our preliminary data on both the penile and vaginal microbiome demonstrates potential value in cases of sexual assault. In this study we describe genital microbial signatures based on the analysis of five male and five female genital samples and compare these results to those from longitudinal studies. Selected taxa, e.g., Gardnerella, Lactobacilli, Finegoldia, Peptoniphilus, and Anaerococci, are shown to be candidate constituents of the genital microbiome that merit investigation for use in sexual assault casework.

BIO-INSPIRED MAGNETIC BEADS FOR ISOLATION OF SPERM FROM HETEROGENOUS SAMPLES IN FORENSIC APPLICATIONS.

Rapid and efficient processing of sexual assault evidence will accelerate forensic investigation and decrease casework backlogs. The standardized protocols currently used in forensic laboratories require the continued innovation to handle the increasing number and complexity of samples being submitted to forensic labs. Here, we present a new technique leveraging the integration of a bio-inspired oligosaccharide (i.e., Sialyl-LewisX) with magnetic beads that provides a rapid, inexpensive, and easy-to-use strategy that can potentially be adapted with current differential extraction practice in forensics labs. This platform (i) selectively captures sperm; (ii) is sensitive within the forensic cut-off; (iii) provides a cost effective solution that can be automated with existing laboratory platforms; and (iv) handles small volumes of sample (∼200 µL). This strategy can rapidly isolate sperm within 25 minutes of total processing that will prepare the extracted sample for downstream forensic analysis and ultimately help accelerate forensic investigation and reduce casework backlogs.

ADAM and ADAMTS gene expression in native and wound healing human lens epithelial cells.

PURPOSE: The ADAMs (a disintegrin and metalloproteinase) and the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin-like motifs) are extracellular proteases that mediate cellular interactions and cell signaling via the modulation of adhesion and the cleavage of cell surface protein ectodomains and extracellular matrix molecules. Gene expression profiling was undertaken to better understand the role of the ADAM and ADAMTS families in the clear native human lenses and following surgical injury with particular relevance to posterior capsule opacification. METHODS: To carry out profile analysis, the lens (t=0d) was dissected into three regions; anterior epithelia, equatorial region, and fiber cells. Capsular bag culture was undertaken as a means of assessing short-term changes (t=6d) and post-cataractous lens capsular bags (ex vivo) were used to predict long-term changes in ADAM/ADAMTS gene expression. RNA was isolated and quantitative real-time (TaqMan) reverse transcription-PCR (RT-PCR) performed. Data were analyzed in terms of cycle threshold number (C(T)) and also normalized relative to endogenous 18S rRNA. RESULTS: High expression of ADAM-9, -10, -15, and -17 was detected in all native lens regions. ADAM-15 expression was a characteristic of the native lens epithelia more than the fibers. Post-surgical injury, lens capsular bags showed a positive shift in ADAM/ADAMTS expression that was significant for ADAM-9, -15, and ADAMTS-3. Ex vivo capsular bags, with a long-term post surgical injury period, maintained high expression of ADAM-9 and -10 genes. CONCLUSIONS: The native human lens expresses ADAM and ADAMTS genes that are differentially regulated following surgical injury. Roles in maintaining cellular adhesion may be of particular importance to native lens tissue integrity and may be lost in the lens wound healing response following cataract surgery.

No neglect-specific deficits in reaching tasks.

It is well established that patients with hemispatial neglect present with severe visuospatial impairments, but studies that have directly investigated visuomotor control have revealed diverging results, with some studies showing that neglect patients perform relatively better on such tasks. The present study compared the visuomotor performance of patients with and without neglect after right-hemisphere stroke with those of age-matched controls. Participants were asked to point either directly towards targets or halfway between two stimuli, both with and without visual feedback during movement. Although we did not find any neglect-specific impairment, both patient groups showed increased reaction times to leftward stimuli as well as decreased accuracies for open loop leftward reaches. We argue that these findings agree with the view that neglect patients code spatial parameters for action veridically. Moreover, we suggest that lesions in the right hemisphere may cause motor deficits irrespective of the presence of neglect and we performed an initial voxel-lesion symptom analysis to assess this. Lesion-symptom analysis revealed that the reported deficits did not result from damage to neglect-associated areas alone, but were further associated with lesions to crucial nodes in the visuomotor control network (the basal ganglia as well as occipito-parietal and frontal areas).

Investigating SNPs flanking the D1S80 locus in a Tamil population from India.

D1S80 is a 16-bp variable number of tandem repeats minisatellite. We analyzed single nucleotide polymorphisms (SNPs) flanking this locus in a Tamil population. Alleles ranged from 15 through 41 repeats, with alleles 18 and 24 being predominant with frequencies of 31% and 34.5%, respectively, suggesting a bimodal allelic distribution. All the 18-repeat alleles are associated with HinfI(+) and FnuAHI(-) restriction site polymorphisms at the 5' and 3' ends, respectively. Allele 24 is associated with HinfI(-) and Fnu4HI(+). Of the alleles tested, 98.5% have a linkage of two specific SNP polymorphisms. If an allele is positive for HinfI, then it is negative for Fnu4HI, and if an allele is negative for HinfI, it is then positive for Fnu4HI, which demonstrates strong linkage disequilibrium between the two polymorphic SNPs. This suggests that reciprocal crossover is not involved in changes in the number of repeats, as few exchanges are seen in the flanking regions. The repeat allele-SNP association might be involved with the internal structure of the locus micropolymorphisms, possibly a double-strand break hotspot.