DNA identification of monozygotic twins.

This study details the differentiation of identical twins based on single mutational base differences. There were three pairs of male monozygotic (MZ) twins in this study. DNA samples from blood, a buccal swab or saliva from each individual were all initially genotyped using 22 autosomal STR and 27 Y-STR loci. Preliminary screening confirmed there were no differences in the STR data between each pair of MZ twins. Whole Genome Sequence (WGS) data were generated from DNA extracted from the three body fluids from each individual. Kinship coefficients with 0.4254, 0.4557 and 0.4543 from 3 twins were generated based on WGS data to further confirm that their relationship was that of MZ twins. The fastq data generated by the Illumina Hiseq 2000 between MZ twins were then treated as "normal" as opposed to "tumor" using commercially available software tools to identify mutational single base changes. Sanger DNA sequencing confirmed there were 1, 5 and 9 single base changes found in WGS data from each of the three MZ twin sets. There was individual variation in the mutational base changes when comparing data from the three body fluids. The methods used in this study to differentiate MZ twins based on WGS data can readily be performed in many operational forensic DNA laboratories using user friendly software.

DNA identification from dental pulp and cementum.

Teeth are one of the body tissues remaining after severe decomposition from which a DNA profile can be obtained to aid in human identification. Currently, the standard approach to isolate DNA from teeth requires pulverizing the entire tooth. This destructive approach compromises any further morphological or anthropological study. We report on two methods of DNA isolation that minimizes destruction of the tooth when accessing the DNA within pulp and cementum. Forty-nine teeth, removed as part of normal dental procedures, were buried for up to 92 days, with a further nine teeth acting as unburied controls. Additionally, four teeth samples collected during a forensic examination were included in this study. The two processes were: using a fine drill to access the pulp from the crown and then using endodontic files to collect the biological material; and using a sterile blade to scrape the cementum. It was found that the samples collected from the cementum had greater DNA quality compared to those samples obtained from the pulp. Microbial activity was found to play a role in the degradation of the nuclear material, reducing DNA yields from pulp. DNA profiling data from 24 loci, including 22 STR markers, indicated that multi-rooted teeth provided better DNA quantity and quality than those with a single root. The DNA quantity obtained from pulp samples of teeth which exhibited cavities was adversely affected, although this DNA loss was not from samples collected from the cementum of teeth in similar condition. Obtaining samples from DNA profiling from the cementum was found to be ideal if the morphological preservation of the tooth is required. Obtaining pathogen DNA is of interest when an occlusal approach to retrieve pulp may serve as a good alternative to prepare DNA without destruction of the tooth structure.

Evaluation of three commercial kits effective identification of menstrual blood based on the D-dimer.

Blood or bloodstains are encountered frequently in forensic investigations. Presumptive and more confirmatory tests for peripheral blood are well established, however, similar methods for menstrual blood identification are less so. D-dimer is a fibrin degradation product that occurs at high concentration in menstrual blood and therefore a potential target to screen for this body fluid. We evaluated three rapid tests to determine if they can discriminate menstrual blood from peripheral remote from a laboratory setting. Their sensitivity, specificity and robustness were also assessed. The assays were: a latex agglutination (Dade Dimertest Latex Assay), SERATEC PMB test and OneStep D-dimer RapidCard InstaTest, both of which are based on lateral flow immunochromatographic analysis. Of the three, greater sensitivity was observed using the OneStep D-dimer RapidCard InstaTest, regardless of whether liquid or a stain was used. This test also detected a result using the smallest volume of menstrual blood, 0.003125 µL. Specificity testing was based on six different body fluids (urine, saliva, peripheral blood, semen, sweats and vaginal fluid) resulting in all 30 samples testing negative for the D-dimer using the OneStep D-dimer RapidCard InstaTest. Mixtures at ratios 1:1, 1:3 and 1:9 (menstrual blood: the other biofluid or PBS) were tested and the results showed that D-dimer could be detected for all samples using either the Dade Dimertest Latex Assay or the OneStep D-dimer RapidCard InstaTest. The body fluids were exposed to environmental stresses such as various temperature (-20 °C, 4 °C, room temperature and 37 °C for 30, 90, 180 and 360 days) and fluctuations in humidity (42%, 76% and 100% humidity at room temperature for 1, 3, 5, 10 and 20 days): all samples were D-dimer positive using the OneStep D-dimer RapidCard InstaTest though the strength decreased relative to the increase of storage time and temperature or humidity. All 6 postmortem blood samples gave a positive result for D-dimer using the OneStep D-dimer RapidCard InstaTest and 2 samples gave a positive response using the Dade Dimertest Latex Assay and the SERATEC PMB test; peripheral blood postmortem samples can show an increase in D-dimer. Menstrual blood was recovered from the pads under the sample wells after testing using the two immunochromatographic assays from which STR alleles could be amplified successfully. The results presented here support the application of these commercial kits for effective identification of menstrual blood.

Y-SNP Haplogroup Hierarchy Finder: a web tool for Y-SNP haplogroup assignment.

The application of massively parallel sequencing (MPS) data from whole genomes has allowed very many more Y-SNP loci to be genotyped simultaneously than previously possible. Although this greatly increases the resolution of Y-SNP haplogroups to link common ancestors, it remains a great challenge to provide a phylogenetic tree to clearly display the relationship of varying haplogroups. Y-SNP Haplogroup Hierarchy Finder is a web tool to generate hierarchical haplogroups based on Y-SNP data with the derived allele at the terminal of a haplogroup tree. The input data can include that from whole-genome sequencing. Confidence in assignment using Y-SNP Haplogroup Hierarchy Finder was demonstrated using Y-SNP genotypes of 1233 samples, sourced from the 1000 genomes project phase 3, used to generate the expected haplogroups. The outcome includes 2 reports: a 'Haplogroup Report' lists mutation types from the submitted Y-SNPs and their corresponding haplogroups, and a 'Haplogroup Hierarchy Report' lists all possible hierarchical haplogroups and ranks the three most supported haplogroups. Each layer of the descending haplogroups from one step to the next is shown and the supporting numbers of Y-SNPs are also included in these reports. All haplogroups that exhibited a clear relationship between the ancestral through to the derived SNPs can be clustered into a hierarchy of haplogroups. The assigned 1233 haplogroups were compared with 2 other software programs designed to assemble haplogroups, which resulted in one where there were many differences and the other one where there was only minor difference. The advantage of this web-based tool is that it provides an easy way to assign Y-SNP haplogroup based on the visualized hierarchical pattern.

A novel co-amplification system for simultaneous amplification of 23 Y-STR and identification of spermatozoa.

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80 ng/0.1 ng) or a mix of the menstrual blood/semen DNA (5 ng/0.1 ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1 ng semen DNA, even in the presence of 5 ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases.

Spermatozoa identification by the 3-plex MSRE-PCR assay: a collaborative exercise.

Identification of semen and spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. In cases of alleged sexual assault where there is a long time gap between the incident and sample collection, or in cases of low sperm count, current methods have limitations of specificity, in the case of presumptive tests for semen, or the problem of recording spermatozoa by microscopy if they are few in number. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay using a spermatozoa-specific DNA methylated marker to identify spermatozoa has been reported previously by our laboratory. A key advantage over current methods is the increased sensitivity and specificity. A transition from a research tool to operational use requires blind trial testing and inter-laboratory trials. We report on a collaborative exercise where reagents of the 3-plex MSRE-PCR were sent to six participating laboratories. Each laboratory used their own equipment, consumables, and the presumptive reagents conventionally for body fluid (such as acid phosphatase or PSA), DNA extraction, and quantification in practical casework. The reagents and protocol for the 3-plex MSRE-PCR assay and 9 samples were provided by the organizing laboratory. The participating laboratories were requested to fill in the questionnaire after testing. The reported results from all the six participating laboratories were concordant and the expected correct results for the presence of spermatozoa. These outcomes verified the reproducibility and feasibility of the 3-plex MSRE-PCR assay. The results also indicated that the 3-plex MSRE-PCR assay was readily accessible to forensic laboratories for integrating it into current forensic casework processes.

Population inference based on mitochondrial DNA control region data by the nearest neighbors algorithm.

Population and geographic assignment are frequently undertaken using DNA sequences on the mitochondrial genome. Assignment to broad continental populations is common, although finer resolution to subpopulations can be less accurate due to shared genetic ancestry at a local level and members of different ancestral subpopulations cohabiting the same geographic area. This study reports on the accuracy of population and subpopulation assignment by using the sequence data obtained from the 3070 mitochondrial genomes and applying the K-nearest neighbors (KNN) algorithm. These data also included training samples used for continental and population assignment comprised of 1105 Europeans (including Austria, France, Germany, Spain, and England and Caucasian countries), 374 Africans (including North and East Africa and non-specific area (Pan-Africa)), and 1591 Asians (including Japan, Philippines, and Taiwan). Subpopulations included in this study were 1153 mitochondrial DNA (mtDNA) control region sequences from 12 subpopulations in Taiwan (including Han, Hakka, Ami, Atayal, Bunun, Paiwan, Puyuma, Rukai, Saisiyat, Tsou, Tao, and Pingpu). Additionally, control region sequence data from a further 50 samples, obtained from the Sigma Company, were included after they were amplified and sequenced. These additional 50 samples acted as the "testing samples" to verify the accuracy of the population. In this study, based on genetic distances as genetic metric, we used the KNN algorithm and the K-weighted-nearest neighbors (KWNN) algorithm weighted by genetic distance to classify individuals into continental populations, and subpopulations within the same continent. Accuracy results of ethnic inferences at the level of continental populations and of subpopulations among KNN and KWNN algorithms were obtained. The training sample set achieved an overall accuracy of 99 to 82% for assignment to their continental populations with K values from 1 to 101. Population assignment for subpopulations with K assignments from 1 to 5 reached an accuracy of 77 to 54%. Four out of 12 Taiwanese populations returned an accuracy of assignment of over 60%, Ami (66%), Atayal (67%), Saisiyat (66%), and Tao (80%). For the testing sample set, results of ethnic prediction for continental populations with recommended K values as 5, 10, and 35, based on results of the training sample set, achieved overall an accuracy of 100 to 94%. This study provided an accurate method in population assignment for not only continental populations but also subpopulations, which can be useful in forensic and anthropological studies.

Identification of spermatozoa using a novel 3-plex MSRE-PCR assay for forensic examination of sexual assaults.

Identification of semen and then spermatozoa is essential to verify that sexual activity has occurred in alleged cases of sexual assault. Microscopic examination commonly used for spermatozoa identification is however time-consuming and can often lead to false-negative results for samples with deformed and, or, limited number of spermatozoa. To address this limitation, we report on a novel 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay to specifically identify spermatozoa. This assay is comprised of 3 markers: a digestive control marker (DC), sperm-specific marker (SP), and Y chromosome marker (SRY). A total of 214 samples from 10 body fluids or tissues were analyzed. Specificity testing showed that all the normal semen samples were unambiguously identified as being sperm-positive, and no other body fluid (or tissues) showed a sperm-specific signal in the electropherogram. Testing for sensitivity showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa by this assay. Mixture analyses illustrated the sensitivity of the assay when the vaginal/semen DNA ratio (80/0.1) was under 800 or the menstrual blood/semen DNA ratio (5/0.1) was under 50, the trace amounts (approximately 0.1 ng) of DNA from semen can still be identified by this 3-plex MSRE-PCR assay. This assay was also applied to the identification of 31 non-probative forensic samples from 18 sexual assault cases. The case studies showed that the 3-plex MSRE-PCR assay was an improvement in the sensitivity of spermatozoa detection.

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

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

A single nucleotide polymorphism panel for individual identification and ancestry assignment in Caucasians and four East and Southeast Asian populations using a machine learning classifier.

Single nucleotide polymorphism (SNP) profiling is an effective means of individual identification and ancestry inferences in forensic genetics. This study established a SNP panel for the simultaneous individual identification and ancestry assignment of Caucasian and four East and Southeast Asian populations. We analyzed 220 SNPs (125 autosomal, 17 X-chromosomal, 30 Y-chromosomal, and 48 mitochondrial SNPs) of the DNA samples from 563 unrelated individuals of five populations (89 Caucasian, 234 Taiwanese Han, 90 Filipino, 79 Indonesian and 71 Vietnamese) and 18 degraded DNA samples. Informativeness for assignment (In) was used to select ancestry informative SNPs (AISNPs). A machine learning classifier, support vector machine (SVM), was used for ancestry assignment. Of the 220 SNPs, 62 were individual identification SNPs (IISNPs) (51 autosomal and 11 X-chromosomal SNPs) and 191 were AISNPs (100 autosomal, 13 X-chromosomal, 30 Y-chromosomal, and 48 mitochondrial SNPs). The 51 autosomal IISNPs offered cumulative random match probabilities (cRMPs) ranging from 1.56 × 10-21 to 3.16 × 10-22 among these five populations. Using AISNPs with the SVM, the overall accuracy rate of ancestry inference achieved in the testing dataset between Caucasian, Taiwanese Han, and Filipino populations was 88.9%, whereas it was 70.0% between Caucasians and each of the four East and Southeast Asian populations. For the 18 degraded DNA samples with incomplete profiling, the accuracy rate of ancestry assignment was 94.4%. We have developed a 220-SNP panel for simultaneous individual identification and ethnic origin differentiation between Caucasian and the four East and Southeast Asian populations. This SNP panel may assist with DNA analysis of forensic casework.

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

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

The detection and identification of saliva in forensic samples by RT-LAMP.

We report on a novel method for saliva identification by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). In our previous report, real-time RT-LAMP was used for blood identification by using HBB detection as a model but in this advanced study, this method was refined for the identification of the more challenging body fluid of saliva. Expression of the18S rRNA gene was used as the internal control and the Statherin (STATH) gene as the saliva-specific marker. A turbidimeter was used for real-time detection of the RT-LAMP products, and confirmation was obtained that the real products were generated using: agarose gel electrophoresis, calcein fluorescence detection and/or enzymatic digestion. The specificity of the test was performed using 42 samples including 7 different body fluids, and the expression of STATH was only observed in all the saliva samples (6) with a threshold time of 39.4 ± 2.9 min. Sensitivity testing showed that RT-LAMP products for STATH were stably detected when the RNA template was not less than 6.25 ng. When the primer concentrations for STATH were two times that of 18S rRNA, saliva could be identified in the body fluid mixtures even at a ratio (saliva:semen) of 1:3 (without loop primer)/1:5 (with loop primer). A multiplex RT-LAMP was established to simultaneously amplify the 18S rRNA and STATH genes, and applied to the identification of saliva on ten non-probative cigarette butts. A positive result for saliva was obtained from all ten butts, even for those that returned a negative or ambiguous result using the amylase test. A direct RT-LAMP test is also reported where the RNA extraction step was omitted to speed the collection of data and all tests using either the simplex or multiplex RT-LAMP resulted in a positive response if saliva was present. Our data provide a simple and effective means to detect the presence of saliva.

A 1204-single nucleotide polymorphism and insertion-deletion polymorphism panel for massively parallel sequencing analysis of DNA mixtures.

Massively parallel sequencing (MPS) technology enables the simultaneous analysis of a huge number of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (indels). MPS also enables the detection of the alleles of minor contributors in a highly unbalanced DNA mixture. In this study, we established a 1204-marker panel optimized for MPS consisting of 987 autosomal markers (964 SNPs and 23 indels), 27 X-chromosome SNPs, 61 Y-chromosome markers (56 SNPs and 5 indels), and 129 mitochondrial SNPs. The DNA samples of six unrelated individuals (two men and four women), 26 nondegraded DNA mixtures (with minor to major ratios of 1:29, 1:39, 1:79, and 1:99), and eight highly artificially degraded DNA mixtures (with minor to major ratios of 1:29, 1:39, 1:79, and 1:99) were analyzed through MPS by using the panel. A scoring system was developed to determine the minor contributors in DNA mixtures based on the genotypes identified using MPS. The genotypes of the 1204 markers were successfully profiled through MPS by using the custom-designed panel. The efficiency of MPS for analyzing these highly degraded samples was lower than that for analyzing nondegraded samples. All minor contributors in the 26 nondegraded and 8 degraded DNA mixtures were accurately assigned using this scoring system based on 964 autosomal SNPs. An association between the observed reads ratio and theoretical ratio of the minor component was noted for nondegraded mixtures. In conclusion, we established a 1204-marker individual identification panel for MPS that successfully analyzed autosomal, X-chromosome, Y-chromosome, and mitochondrial SNPs and indels simultaneously. In combination with the newly developed scoring system, the panel can accurately identify minor contributors in nondegraded and highly degraded DNA mixtures.

Establishment of 11 linked X-STR loci within 1.1 Mb to assist with kinship testing.

This report identifies and characterizes 10 novel short tandem repeat (STR) loci on the human X chromosome, all of which are within a range of 1.1 Mb. These newly characterized loci were developed to aid in kinship assignment when the X chromosome is specifically required. The repeat DNA sequences were identified initially using data in GenBank and are located immediately upstream and downstream from the previously described locus DXS6807. Only those loci with seven or more observed alleles were used for further study resulting in the identification of 10 new loci. The distance between each pair of loci ranged from 24,998 to 244,701 bp with an average of approximately 110.8 kb. The number of observed alleles ranged from 7 to 30 for these 10 loci with a polymorphic information content ranging from 0.593 to 0.930. The LOD score from a pairwise linkage study ranged from 4.40 to 23.73, indicating that these 11 loci were highly linked, as expected. In line with standard forensic practice, all 11 loci can be amplified in one multiplex reaction, and comprehensive allelic ladders for all the loci have been constructed. These newly established 11 linked STR loci on the human X chromosome were found to be highly polymorphic and have the potential to aid in kinship testing where the X chromosome loci currently plays a role.

Investigation of length heteroplasmy in mitochondrial DNA control region by massively parallel sequencing.

Accurate sequencing of the control region of the mitochondrial genome is notoriously difficult due to the presence of polycytosine bases, termed C-tracts. The precise number of bases that constitute a C-tract and the bases beyond the poly cytosines may not be accurately defined when analyzing Sanger sequencing data separated by capillary electrophoresis. Massively parallel sequencing has the potential to resolve such poor definition and provides the opportunity to discover variants due to length heteroplasmy. In this study, the control region of mitochondrial genomes from 20 samples was sequenced using both standard Sanger methods with separation by capillary electrophoresis and also using massively parallel DNA sequencing technology. After comparison of the two sets of generated sequence, with the exception of the C-tracts where length heteroplasmy was observed, all sequences were concordant. Sequences of three segments 16184-16193, 303-315 and 568-573 with C-tracts in HVI, II and III can be clearly defined from the massively parallel sequencing data using the program SEQ Mapper. Multiple sequence variants were observed in the length of C-tracts longer than 7 bases. Our report illustrates the accurate designation of all the length variants leading to heteroplasmy in the control region of the mitochondrial genome that can be determined by SEQ Mapper based on data generated by massively parallel DNA sequencing.

A panel of 130 autosomal single-nucleotide polymorphisms for ancestry assignment in five Asian populations and in Caucasians.

Ancestry informative single-nucleotide polymorphism (AISNP) panels for differentiating between East and Southeast Asian populations are scarce. This study aimed to identify AISNPs for ancestry assignment of five East and Southeast Asian populations, and Caucasians. We analyzed 145 autosomal SNPs of the 627 DNA samples from individuals of six populations (234 Taiwanese Han, 91 Filipinos, 79 Indonesians, 60 Thais, 71 Vietnamese, and 92 Caucasians) using arrays. The multiple logistic regression model and a multi-tier approach were used for ancestry classification. We observed that 130 AISNPs were effective for classifying the ethnic origins with fair accuracy. Among the 130 AISNPs, 122 were useful for stratification between these five Asian populations and 64 were effective for differentiating between Caucasians and these Asian populations. For differentiation between Caucasians and Asians, an accuracy rate of 100% was achieved in these 627 subjects with 50 optimal AISNPs among the 64 effective SNPs. For classification of the five Asian populations, the accuracy rates of ancestry inference using 20 to 57 SNPs for each of the two Asian populations ranged from 74.1% to 100%. Another 14 degraded DNA samples with incomplete profiling were analyzed, and the ancestry of 12 (85.7%) of those subjects was accurately assigned. We developed a 130-AISNP panel for ethnic origin differentiation between the five East and Southeast Asian populations and Caucasians. This AISNP set may be helpful for individual ancestral assignment of these populations in forensic casework.

SEQ Mapper: A DNA sequence searching tool for massively parallel sequencing data.

The development of massively parallel sequencing (MPS) has increased greatly the scale of DNA sequencing. The analysis of massive data-files from single MPS analysis can be a major challenge if examining the data for potential polymorphic loci. To aid in the analysis of both short tandem repeat (STR) and single nucleotide polymorphisms (SNP), we have designed a new program called SEQ Mapper to search for genetic polymorphisms within a large number of reads generated by MPS. This new program has been designed to perform sequence mapping between reference data and generated reads. As a proof-of-concept, sequences derived from the allelic ladders of five STR loci and data from the amelogenin locus were used as reference data sets. Detecting and recording the polymorphic nature of each STR loci was performed using four levels of search criteria: the entire STR locus spanning the two primers; the STR region plus the two primer sequences; the STR region only; and the two primers only. All the genotypes of 5 STR loci and the amelogenin gene were identified correctly using SEQ Mapper when compared to results obtained from capillary electrophoresis based on 10 test samples in this study. SEQ Mapper is a useful tool to detect STR or SNP alleles generated by MPS in both clinical medicine and forensic genetics.

Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system.

The identification of a specific body fluid encountered in a forensic investigation can give crucial information. This identification can be aided by methylation profiles based on selected markers specific to a range of biofluids. In this study, the open database of Infinium HumanMethylation450 BeadChip was searched for markers specific for semen, vaginal fluids, saliva, venous blood and menstrual blood. A total of 8 biofluid-specific methylated markers and 2 control markers were combined into a 10-plex methylation sensitive restriction enzyme-PCR (MSRE-PCR) system. Based upon the analysis of 100 DNA samples from these 5 biofluid types, unambiguous results were obtained to identify the body fluid from which it originated. Validation studies of the developed 10-plex MSRE-PCR included sensitivity, reproducibility and mixed body fluids. Co-amplification of the established MSRE-PCR system and the microsatellite loci in AmpFlSTR® MiniFiler™ PCR Amplification Kit was performed to generate both the methylation profile for biofluid type and the miniSTR profile. This allowed human identification and the identification of the body fluid type to be performed in a single reaction. The results of this study displayed the applicability of this 10-plex MSRE-PCR system in forensic science.

Species Determination: The Role and Use of the Cytochrome b Gene.

The illegal trade in ivory and rhino horn has led to a catastrophic decline in elephant and rhino populations worldwide. These iconic high profile species are one part of the illegal trade in wildlife products that threatens these species and the ecological balance in the habitats in which they live. Identification of the species present is required to determine that the trade is illegal and contrary to national legislation. This chapter details a robust DNA technique using part of the cytochrome b gene on the mitochondrial genome that will work on poor quality samples such as powdered horn or ivory products including statues and carvings. An appropriate DNA extraction technique is required to obtain at least 1 ng of DNA from which the amplification of part of the cytochrome b gene using universal primers is performed. This produces a fragment of 486 bp in size which can be sequenced using standard technologies. The resulting sequences are then aligned to voucher specimens or sequences on reliable databases. Analyses of the data should lead to confident species identification.

Adult femicide victims in forensic autopsy in Taiwan: A 10-year retrospective study.

BACKGROUND: Violence against women and adult femicides are critical medico-legal issues worldwide. Intimate partner violence is one of the leading contributory risk factors. This study aimed to describe the characteristics of femicides in Taiwan. METHODS: A retrospective analysis of forensic autopsy records of adult femicide victims in Taiwan during a 10-year period was carried out. The age, victim-offender relationship, injury patterns and causes of death were analyzed. RESULTS: Among the 220 adult femicide victims recruited, 114 were killed by intimate partners and 106 were killed by non-intimate partner offenders. The average age of victims killed by intimate partners (40.0 y/o) were younger than those killed by non-intimate partner perpetrators (48.6 y/o). The most common site of injuries in the intimate partner group and the non-intimate partner group was the neck and the upper limbs, respectively. The rates of bruise and intracranial injury of non-intimate partner group were significantly higher than that of the intimate partner group. The most common causes of death in both groups were strangulation and sharp force injury. The heart injury was significantly more frequent in victims offended by intimate partners than by other assailants. CONCLUSIONS: The characteristics of adult femicides, and the patterns of injury in victims killed by intimate partners and non-intimate partner offenders were different. This data is helpful for corpus inspection in forensic casework and for strategic planning of femicides prevention.