Search

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

An increasing number of studies on massively parallel sequencing of mitochondrial DNA (mtDNA) have been reporting identification of various types of noise or off-target sequences. Herein, we report that an off-target haplotype (sequence length 192 bp) observed in MiSeq data of mtDNA at nucleotide position 16,209-16,400 was likely caused by polymorphic nuclear mitochondrial DNA sequences (NumtS). Buccal DNA samples from Volunteers #001-004 and Control DNA 007 were amplified with our multiplex system of the B (15,998-16,172), C (16,209-16,400), and E (30-289) regions using 2000 copies of mtDNA. A sample index was added using a Nextera XT index kit, and MiSeq Reagent Nano Kit v2 was used in 2 × 251 cycles on a MiSeq FGx. FASTQ files were analyzed by CLC Genomics Workbench 21.0.3. The extracted SAM files were analyzed using our original Excel macro to sum up the read counts as the phased variant calls for each region. An off-target haplotype differing at 19 sites against the revised Cambridge reference sequence was observed in Volunteer #001 (4 in 10 MiSeq data), Volunteer #002 (2 in 9), and Control DNA 007 (6 in 9). In a BLAST search, the sequence of the off-target haplotype matched perfectly to three polymorphic NumtS (Poly_NumtS_430 [KM281528.1], HSA_NumtS_587 [HE613849.1], and nuclear fossil [S80333.1]) and BAC clone of chromosome 11 (AC107937.2). The sequence also matched perfectly to a Filipino mtDNA (KC993973.1) which was inferred as a chimeric sequence of mtDNA and the HSA_NumtS_587. The sequence of the off-target haplotype was not contained in the latest human reference genome sequence (GRCh38.p13). In a phylogenetic tree, the off-target haplotype was genetically distant from modern human mtDNA and not directly connected to them. In conclusion, observed off-target haplotype amplified by our multiplex system was likely caused by Poly_NumtS_430 or HSA_NumtS_587.

In forensic mitochondrial DNA (mtDNA) analysis, quantitative PCR (qPCR) is usually performed to obtain high-quality sequence data for subsequent Sanger or massively parallel sequencing. Unlike methods for nuclear DNA quantification using qPCR, a calibrator is necessary to obtain mtDNA concentrations (i.e., copies/µL). Herein, we developed and validated a mtDNA quantification method based on a SYBR Green assay by following MIQE [Bustin et al., Clin. Chem. 55 (2009) 611-22] and other guidelines. Primers were designed to amplify nucleotide positions 16,190-16,420 in hypervariable region 1 for qPCR using PowerUp SYBR Green and QuantStudio 5. The optimized conditions were 0.3 µM each primer and an annealing temperature of 60 °C under a 2-step cycling protocol. K562 DNA at 100 pg/µL was converted into a mtDNA concentration of 16,400 copies/µL using linearized plasmid DNA. This mtDNA calibrator was obtained by cloning the synthesized DNA fragments of mtDNA (positions 16,140-16,470) containing a 100-bp inversion. The linear dynamic range of the K562 standard curve was 10,000-0.1 pg/µL (r

Forensic human identification (HID) laboratories occasionally encounter non-specific peaks generated by non-human DNA. Casework samples for human short tandem repeat (STR) profiling may be contaminated by animal DNA because of the specific environment or situation from which they were obtained. Validation studies for HID kits have reported that non-specific peaks generated from some animals are observed near the human amelogenin peak. In this study, we first revealed that DNA sequences associated with the non-specific peaks generated from animal DNA differ from one animal family to the other. However, non-specific peaks cannot be analyzed using the remainder of polymerase chain reaction (PCR) products left over from conventional HID kits when human and animal DNA are mixed. To overcome this issue, we have developed a novel analysis method of using non-specific peaks generated from animal DNA in human STR profiling to identify the source of contaminating animal DNA at the family level. The method applied here is termed as blocking PCR, which involves selective animal DNA re-amplification by blocking nontarget human amelogenin DNA amplification using an oligonucleotide probe that specifically binds to human amelogenin using the remaining PCR product from the HID kit. Our data demonstrated that HID and family discrimination among animals that are often encountered in forensic contexts could be performed simultaneously. This study enabled recovery of more information from limited quantities of casework samples contaminated with animal DNA, which would be useful for forensic HID scientists.

Rapid identification of pathogenic agents is important in response to the emergence of biocrime and bioterrorism, to facilitate appropriate confinement and treatment. As the rapid determination system of viral genome sequences (RDV method) using exhaustive gene amplification is useful for rapid identification, we examined whether this method could be applied to forensic samples. To detect pathogenic virus in a cat with suspected viral infections, fluid swab samples were applied to the RDV method. The following steps were performed: viral propagation, extraction of the viral genome, amplification of the first library, fragmentation of the library, amplification of the second library using non-specific primer sets, and direct sequencing of the amplicon. To confirm the viruses detected by this method, we performed conventional PCR using virus-specific primers. We detected pathogenic virus genome sequences from the swab samples and confirmed infection with these viruses. In addition, we directly detected a viral genome sequence from the nasal swab sample without the viral propagation step. The RDV method is infrequently used in forensic analysis. This method is practicable with equipment existing in a normal laboratory and is useful for rapid detection and identification of pathogenic viruses in forensic samples. This method would also be applicable to the detection of bacteria and fungi.

In short tandem repeat (STR) analysis, length polymorphisms are detected by capillary electrophoresis (CE). At most STR loci, mobility shift due to sequence variation in the repeat region was thought not to affect the typing results. In our recent population studies of 1501 Japanese individuals, off-ladder calls were observed at the D12S391 locus using PowerPlex Fusion in nine samples for allele 22, one sample for allele 25, and one sample for allele 26. However, these samples were typed as ordinary alleles within the bins using GlobalFiler. In this study, next-generation sequencing analysis using MiSeq was performed for the D12S391 locus from the 11 off-ladder samples and 33 other samples, as well as the allelic ladders of PowerPlex Fusion and GlobalFiler. All off-ladder allele 22 in the nine samples had [AGAT]11[AGAC]11 as a repeat structure, while the corresponding allele was [AGAT]15[AGAC]6[AGAT] for the PowerPlex Fusion ladder, and [AGAT]13[AGAC]9 for the GlobalFiler ladder. Overall, as the number of [AGAT] in the repeat structure decreased at the D12S391 locus, the peak migrated more slowly using PowerPlex Fusion, the reverse strand of which was labeled, and it migrated more rapidly using GlobalFiler, the forward strand of which was labeled. The allelic ladders of both STR kits were reamplified with our small amplicon D12S391 primers and their mobility was also examined. In conclusion, off-ladder observations of allele 22 at the D12S391 locus using PowerPlex Fusion were mainly attributed to a relatively large difference of the repeat structure between its allelic ladder and off-ladder allele 22.

Phenomena called allele dropouts are often observed in crime stain profiles. Allele dropouts are generated because one of a pair of heterozygous alleles is underrepresented by stochastic influences and is indicated by a low peak detection threshold. Therefore, it is important that such risks are statistically evaluated. In recent years, attempts to interpret allele dropout probabilities by logistic regression using the information on peak heights have been reported. However, these previous studies are limited to the use of a human identification kit and fragment analyzer. In the present study, we calculated allele dropout probabilities by logistic regression using contemporary capillary electrophoresis instruments, 3500xL Genetic Analyzer and 3130xl Genetic Analyzer with various commercially available human identification kits such as AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. Furthermore, the differences in logistic curves between peak detection thresholds using analytical threshold (AT) and values recommended by the manufacturer were compared. The standard logistic curves for calculating allele dropout probabilities from the peak height of sister alleles were characterized. The present study confirmed that ATs were lower than the values recommended by the manufacturer in human identification kits; therefore, it is possible to reduce allele dropout probabilities and obtain more information using AT as the peak detection threshold.

A 64-year-old male came to our hospital emergency department with fever and consciousness disturbance. Culture tests of blood and spinal fluid samples revealed meningococci (Neisseria meningitidis), and we made a diagnosis of meningococcal meningitis. Brain magnetic resonance imaging (MRI) findings revealed ventriculitis. Ceftriaxone was administered for 17 days, however, relapse was noted after that was discontinued, with neutropenia and renal impairment thought to be adverse reactions to the beta-lactam antibiotic. Hence, treatment was switched to oral administration of moxifloxacin for a total of 12 weeks, including in an outpatient setting. After moxifloxacin was discontinued, no side effects or relapse were seen, and treatment was ended. Although antibacterial agents generally show favorable effects for meningococcal meningitis, we consider that sufficient antimicrobial therapy is difficult in cases complicated with ventriculitis.

A method based on liquid chromatography-electrospray mass spectrometric analysis of the enzymatically digested amplicons derived from the mitochondrial 16S rRNA gene was established for the discrimination of toxic pufferfish. A MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q Exactive mass spectrometer (Thermo Fisher) were employed for separation and detection, respectively. Monoisotopic masses of the oligonucleotides were calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm on average) and precision (relative standard deviation, 0.49 ppm on average) were achieved, and a mass accuracy of

Allele frequencies for 22 autosomal short tandem repeat loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, and D22S1045) were obtained from 1501 unrelated individuals sampled from the Japanese population.

An experimental DNA extraction kit (new kit) was recently developed to extract DNA from degraded skeletal remains without the need for powdering the samples. We compared the utility of the new kit with the conventional phenol/chloroform method using real-time quantitative PCR and multiplex STR analysis. The new kit yielded large amounts of DNA from a compact bone fragment compared with the conventional phenol/chloroform method. We were able to extract sufficient DNA for STR analysis from 75% (3 of 4) and 60% (3 of 5) of the un-powdered tooth and bone samples, respectively, using the new kit. We were able to perform mini-STR analysis of the remaining samples using DNA extracted with the new kit. Furthermore, we successfully performed mitochondrial DNA sequencing of every sample. The new kit simplifies the DNA extraction procedure as it does not require powdering samples. Decreasing the number of procedural steps in DNA extraction will be beneficial in controlling DNA contamination in laboratories. Our results suggest that the new kit may be used for the simple, simultaneous extraction of DNA from multiple samples.

Information regarding the ancestral and geographical origins of biological evidence samples may be useful for crime investigators as they narrow down the possible donors of the sample. A method for simultaneous analysis of seven biallelic markers (M130, M131, M57, M125, M175, M122 and M134) was developed for forensic application. M57, M125 and M131 are included to identify haplogroups inferred as having originated in the Japanese archipelago. Our method employs allele-specific PCR and fragment analysis using fluorescently labeled primers and capillary electrophoresis. This method can be used to assign a haplogroup from both of degraded male DNA samples and DNA samples containing a mixture of female and male DNA by designing PCR primers that generate small amplicons and are highly specific for targets on the Y chromosome. A total of 1346 samples from Japanese males collected from the four major islands and Okinawa island were classified into seven Y binary haplogroups i.e., C-M130, C-M131, D-M57, D-M125, O-M175, O-M122 and O-M134, and a "no-mutation detected" group and their frequencies were 0.0617, 0.0565, 0.1441, 0.182, 0.3418, 0.11, 0.0847 and 0.0193, respectively. Samples of "no-mutation detected" were further analyzed by direct sequencing for identification of the major haplogroup to which they belong. Along with the haplogroup data, we report haplotype data for the 16 Y-STR markers included in the AmpFlSTR ® Yfiler™ PCR amplification kit (Applied Biosystems). These data will be useful in the prediction of haplogroups based on Y-STR haplotypes.

We and others have previously shown that reactive microglia express the major histocompatibility complex (MHC) class I and class II antigens in the hippocampus of patients suffering from epilepsy. Although the MHC glycoproteins serve as restriction elements for T lymphocytes, there is little information available regarding T lymphocytes in hippocampal sclerosis. In the present study, we investigated T lymphocyte infiltration in human hippocampi in four cases of epilepsy with hippocampal sclerosis, as well as in four control cases without neurological disease. No CD8- or CD4-positive T lymphocytes were seen in hippocampi from the control cases. In contrast, CD8- and CD4-positive T lymphocytes had infiltrated into the hippocampi of patients with hippocampal sclerosis. In addition, expression of intercellular adhesion molecule-1 was diffusely upregulated in the hippocampi with hippocampal sclerosis. These results indicate that T lymphocyte infiltration is involved in the pathology of hippocampal sclerosis.

Short tandem repeat studies are powerful tools for parentage analysis and for identification of missing persons, victims of murder, and victims of mass fatalities when reference samples are unavailable. The primer in the Identifiler® kit failed to amplify an allele at the D19S433 locus, producing a silent (“null”) allele. The causal mutation is a base change (G>A) 32 nucleotides downstream from the 3′ end of the AAGG repeats. The silent alleles are problematical in parentage analysis because when transmitted, they can cause a parent–child inconsistency that is unrelated to Mendelian genetics. The inconsistency is sometimes termed an “apparent opposite homozygosity” and it produces false evidence of nonparentage. Alternative primers were designed to amplify the D19S433 locus alleles and they detect the silent allele. Frequencies of the (no longer) silent allele were determined to be 0.0114 in 176 people from Shizuoka (Honshu) and 0.0128 in 156 people from Okinawa.

A total of 1079 Japanese males were typed for the following 16 Y chromosomal short tandem repeat (Y-STR) markers: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448 using an AmpFlSTR® Yfiler™ PCR Amplification kit (Applied Biosystems). A total of 950 haplotypes for the 16 Y-STR markers were detected and, of these, 886 haplotypes were unique. The most frequent haplotype was found in 22 Japanese males. The haplotype diversity was 0.9992, indicating a high potential for differentiating between male individuals. There were 10 haplotypes with no allele detected at the DYS448 marker. Thus, the presence of such atypical haplotypes should be noted, when DNA typing results obtained from degraded DNA samples and/or DNA mixture samples from more than one male individual are being interpreted.

Short tandem repeat (STR) typing is widely used in forensic investigation. When the same DNA sample is analyzed with different STR typing kits, a typing discrepancy is occasionally observed. In this study, we examined the cause of a typing discrepancy in a sample at D5S818 locus. This sample was designated as 10, 12 using Identifiler(®) , Identifiler(®) Plus, GlobalFiler(®) , PowerPlex(®) 16HS, and PowerPlex(®) 18D, but as 9.3, 12 using PowerPlex(®) Fusion. Sequencing results indicated that the shorter allele in the sample had a deletion (U31Tdel) at 31 nucleotides upstream of the repeat region (AGAT)10 . This deletion was located in the binding site of the published D5S818 forward primer in PowerPlex(®) 16 and was only 9 and 11 nucleotides downstream of our estimated 5' end position of D5S818 forward primer in GlobalFiler(®) and PowerPlex(®) 18D, respectively. We also examined the effect of primer length on the heterozygous peak balance in this sample.

This study is concerned with the demand for ductility of beams in steel moment-resistant frames. Numerical response analysis was carried out for 15 frames against a variety of ground motions. This paper describes the magnitude of plastic deformation introduced into beam-ends. The purpose of this study is to deduce the demand for ductility of beams. Maximum plastic rotation, maximum increment of plastic rotation during a half-cycle of vibration, and the range of variable plastic rotation are considered as the parameters that represent the magnitude of plastic deformation. The results are summarized as formulas to predict those parameters based on maximum story drift angles., Shanghai, China, 13-15 June, 2005

In this study, DNA was extracted using an AutoMate Express™ and an EZ1 Advanced XL from liquid blood, fresh and aged bloodstains, and fresh and aged semen stains. Extracted DNA was quantified by real-time PCR using the D17Z1 locus. Short tandem repeat typing was performed using an AmpFlSTR(®) Identifiler kit. The yields of DNA obtained by the AutoMate Express™ were higher from fresh bloodstains and fresh semen stains, almost the same from aged bloodstains and aged semen stains, but slightly lower from liquid blood compared with those obtained by the EZ1 Advanced XL. The addition of dithiothreitol or the use of PrepFiler™ lysis buffer improved the EZ1 Advanced XL results from fresh bloodstains, but not for liquid blood and aged bloodstains. Our results demonstrated that the PrepFiler™ lysis buffer is the main contributor to the higher DNA yields of the AutoMate Express™ for fresh bloodstains.

Peak Particle Velocity (PPV) of ground vibration induced by blasting have been used for long time as a standard for evaluating damage of structures. However, in recent years, it is often discussed whether the PPV standard is adequate or not for evaluating public environmental disturbances. In such case, Vibration Level (VL) is used, which is established and defined to allow for the feeling of human body against the acceleration of vibration. Hence the necessity of measurement by both PPV and VL is increasing. If the relation between PPV and VL can be quantitatively clarified, then it is possible to estimate PPV from VL and vice versa. The relation, however, is complicated in case of ground vibration induced by practical delayed blastings because of some factors, namely such as the frequency components and the shape of wave form.In this paper, firstly, a new method to calculate VL from the velocity wave form was discussed. Through the discussion, many factors were investigated which have to be considered when the vibration wave form is converted into VL. Then such method was applied to measured waves caused by tunnelling blasting in order to evaluate a variation due to each primary factor. As a result, a new relationship equation between PPV and VL for ground vibration due to blasting was proposed.

Performing short tandem repeat (STR) analysis from degraded DNA is a challenge for forensic biologists. For assessing the quality and quantity of DNA, we developed quantitative PCR assays to determine the extent of DNA degradation. Quantitative PCR assays using primers that generate two sizes of amplicons from the same region of genomic DNA were used to determine the extent of DNA degradation. These quantitative PCR assays were used with artificially degraded DNA and degraded DNA extracted from aged bloodstains. Increased DNA degradation correlated with a decrease in the number of detectable loci in STR analysis. The extent of DNA degradation and the number of loci detected by STR analysis varied depending on the method of degradation. The extent of degradation of DNA extracted from aged bloodstains correlated well with that of DNA artificially degraded by DNase I in the presence of Mn(2+). Thus, determination of the extent of DNA degradation was helpful for estimating the number of detectable loci. Furthermore, this estimation method is expected to save time and labor, and is particularly suitable when only a limited amount of DNA can be extracted from casework samples.

We have developed a multiplex, single nucleotide polymorphism (SNP) typing system for forensic identification, based on the Invader assay. Using this system, fluorescence data for 21 SNP loci were collected and analyzed. We used endpoint genotyping, commonly used with the Invader assay, and also developed more reliable typing criteria because endpoint typing was occasionally unable to clearly identify the genotype of some loci. Analysis of the fluorescence data identified criteria that had high reproducibility for each genotype. One such criterion is the climbing angle of the curve resulting from two-dimensional plots of the two kinds of fluorescence used for the Invader assay. The climbing angle was observed at the time during the reaction when either or both of the fluorescence intensities increased most significantly. The angles were remarkably associated with homozygous genotypes. On the other hand, all heterozygote endpoint fluorescence ratios were highly reproducible and had very little aberration. These values enabled SNP typing to be more clearly defined compared with typing using only the endpoint fluorescence ratio, which is commonly used with the Invader assay. The values suggested in this study are easily calculated from raw fluorescence data and will be useful for multiplex SNP typing based on the Invader assay.

AlAs/Al/AlAs heterostructures are fabricated on (001)GaAs by molecular beam epitaxy (MBE) and migration enhanced epitaxy (MEE). The Al and successive AlAs layers are grown at room temperature by MBE and MEE, respectively, while the underlying AlAs layer is grown at 600°C by MBE. Single crystal Al layers are grown successfully. The epitaxy relationship of Al with the underlying AlAs layer is (011)[100]Al‖(001)[110]AlAs. Detailed RHEED analysis elucidates the lattice relaxation processes of Al on AlAs as well as the growth process. It is suggested that the initially deposited 2(1) ML thick Al layers occupy sublattice sites in the AlAs lattice with the same lattice spacing as AlAs on As- (Al-) stabilized AlAs and that the lattice relaxation and the formation of the fcc Al lattice is initiated during deposition of the 3rd (2nd) layer on As- (Al-) stabilized AlAs. The top AlAs layer is single crystalline and grows in an island growth mode. The observed epitaxy relationship of AlAs on Al is (001)[110]AlAs‖(011)[100]Al.

Typing of single nucleotide polymorphisms (SNPs) has advantages in forensic DNA identification because SNPs are abundant in genomic DNA and are easily detected using an automated high-throughput system. In this study, we examined the effectiveness of an automated multiplex SNPs typing system based on the "Invader assay". Using this system, multiplex SNPs typing is completed in 40 min without any complex procedures. The typing results were confirmed by direct sequencing, and no inconsistencies were detected between the sequencing results and the typing results. The population data of 21 SNPs from 113 Japanese indicates that the matching probability is about 1.3 x 10(-9). In our experiment, all of 21 SNPs were correctly detected from degraded samples from which typing of short tandem repeat markers was difficult.

When mitochondrial DNA (mtDNA) heteroplasmies are detected, they often confound forensic identification, especially if they are the result of poor biological sampling. In this study, we determined the ratio of heteroplasmy in samples that were amplified from a very small amount of template mtDNA or a few cells using a highly sensitive nested polymerase chain reaction (PCR) procedure and a direct sequencing analysis. As a result, more than half of the detected sequences (i.e., 17/20, 15/20, and 14/20) showed homoplasmy derived from a variation in the heteroplasmy proportion when only 10 copies of template mtDNA samples were amplified and analyzed. Additionally, with products amplified from one or several white blood cells (WBCs), several previously undetected heteroplasmies were detected. These results indicate the risks associated with using highly sensitive mtDNA techniques in forensic investigations because of the variable proportions of heteroplasmy or nucleotide substitutions that can possibly be detected from a very small biological sample.

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were determined for 1204 unrelated Japanese individuals. A total of 741 different mtDNA haplotypes were found, 157 of which were seen in multiple individuals. Twenty-seven of these individuals showed point heteroplasmy. The most frequent haplotype (16223T-16362C-73G-263G-315.1C) was found in 31 individuals and the second most frequent haplotypes (16129A-16223T- 16362C-73G-152C-263G-309.1C-315.1C) was found in 24 individuals. The haplotypes diversity and random match probability were calculated to be 0.9969 and 0.0040, respectively.

The molecular beam epitaxial growth processes of Al single‐crystal layers deposited at room temperature on As‐stabilized and on Al‐stabilized AlAs surfaces are investigated in situ using reflection high‐energy electron diffraction (RHEED). The epitaxy relationship of Al with the underlying AlAs is (011)[100]Al//(001)[110]AlAs irrespective of the surface stoichiometry of AlAs. Detailed RHEED investigation suggests that the initially deposited two one‐monolayer‐thick Al layers form a sphalerite lattice on As‐(Al‐)stabilized AlAs with the same lattice spacing as AlAs, while the deposition of additional Al layers induces the structural phase transition from the sphalerite lattice to an fcc lattice with bulk Al lattice constant. Thick epitaxial Al layers show (4×1) surface reconstruction.