Development and validation of a SYBR green-based mitochondrial DNA quantification method by following the MIQE and other guidelines.

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.

Poly_NumtS_430 or HSA_NumtS_587 observed in massively parallel sequencing of the mitochondrial HV1 and HV2 regions.

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.

Analysis Comparison for Rapid Identification of Pathogenic Virus from Infected Tissue Samples.

When examining infectious samples, rapid identification of the pathogenic agent is required for diagnosis and treatment or for investigating the cause of death. In our previous study, we applied exhaustive amplification using non-specific primers (the rapid determination system of viral genome sequences, the RDV method) to identify the causative virus via swab samples from a cat with a suspected viral infection. The purpose of the current study is to investigate suitable methods for the rapid identification of causative pathogens from infected tissue samples.

Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.

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.

Non-specific peaks generated by animal DNA during human STR analysis: Peak characteristics and a novel analysis method for mixed human/animal samples.

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.

Next-generation sequencing analysis of off-ladder alleles due to migration shift caused by sequence variation at D12S391 locus.

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.

[A case of meningococcal meningitis that was difficult to treat owing to concurrent ventriculitis].

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.

D5S818 Typing Discrepancy Between PowerPlex(®) Fusion and Other STR Kits Including GlobalFiler(®) Caused by a One-base Deletion in 31 Nucleotides Upstream of the Repeat Region.

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.

Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP Products As Determined by Liquid Chromatography/Quadrupole-Orbitrap Hybrid Mass Spectrometry.

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.

Allele frequencies for 21 autosomal short tandem repeat loci obtained using GlobalFiler in a sample of 1501 individuals from the Japanese population.

Allele frequencies for 21 autosomal short tandem repeat loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, and D2S1338) were obtained using the GlobalFiler kit from 1501 unrelated individuals sampled from the Japanese population.

Estimating allele dropout probabilities by logistic regression: Assessments using Applied Biosystems 3500xL and 3130xl Genetic Analyzers with various commercially available human identification kits.

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.

Allele frequencies for 22 autosomal short tandem repeat loci obtained by PowerPlex Fusion in a sample of 1501 individuals from the Japanese population.

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.

A comparison of DNA extraction using AutoMate Express™ and EZ1 advanced XL from liquid blood, bloodstains, and semen stains.

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.

Estimation of the detection rate in STR analysis by determining the DNA degradation ratio using quantitative PCR.

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.

Infiltration of T lymphocytes and expression of icam-1 in the hippocampus of patients with hippocampal sclerosis.

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.

Criterion values for multiplex SNP genotyping by the invader assay.

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.

A forensic method for the simultaneous analysis of biallelic markers identifying Y chromosome haplogroups inferred as having originated in Asia and the Japanese archipelago.

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.

Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method.

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.

Automated SNPs typing system based on the Invader assay.

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.

A D19S433 primer binding site mutation and the frequency in Japanese of the silent allele it causes.

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.

Mitochondrial DNA population data of HV1 and HV2 sequences from Japanese individuals.

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.