A highly efficient scheme for library preparation from single-stranded DNA.

Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA.

Three Autopsy Cases of Non-Meningococcal Waterhouse-Friderichsen Syndrome with Hypoplastic Spleen or Post-Splenectomy Status.

We report three cases of Waterhouse-Friderichsen syndrome (WFS) that were confirmed during forensic autopsies. Case 1 involved a man in his 50s post-splenectomy. Bacteriological examination revealed Streptococcus pneumoniae (S.

Allele frequencies of 31 autosomal short tandem repeat (auSTR) loci obtained using the Precision ID GlobalFiler™ NGS STR Panel v2 in 322 individuals from the Japanese population.

In human identification methods that target short tandem repeats (STRs), massively parallel sequencing (MPS) technology has made it possible to genotype at the level of the specific sequence itself. This allows for the detection of repeat unit variants and single nucleotide polymorphisms (SNPs) adjacent to the STRs. Using the GlobalFiler™ NGS STR Panel v2, Ion S5, and Converge software, this study constructed a Japanese database of 31 autosomal STRs (auSTRs) and two sex markers from 322 individuals.

Highly sensitive sex determination method using the exon 1 region of the amelogenin gene.

Sex determination is a crucial factor in the identification of unidentified human remains. Sex determination by DNA analysis is particularly useful because it can be applied to samples for which morphological characteristics are unavailable. Because samples handled in forensic DNA typing are easily degraded by environmental factors and microorganisms, there is a need for a method that can accurately determine sex even in highly decayed samples.

Detection of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in blood in a forensic case using liquid chromatography-electrospray ionization linear ion trap mass spectrometry.

We present a case of fatal poisoning by 4-F-methcathinone (4-FMC; also called flephedrone), 4-methoxy-α-pyrrolidinopentiophenone (4-MeO-α-PVP), 4-fluoro-α-pyrrolidinopentiophenone (4-F-α-PVP), and α-pyrrolidinohepatanophenone (PV8). In this study, we compared the mass spectra of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, PV8, and α-pyrrolidinohexanophenone between LC-ESI-LIT-MS and GC-EI-MS analyses. Subsequently, we applied LC-ESI-LIT-MS for detection and quantification analyses of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in human authentic whole blood samples.

Postmortem distribution of mepirapim and acetyl fentanyl in biological fluid and solid tissue specimens measured by the standard addition method.

Purpose: Mepirapim is a new synthetic cannabinoid. We previously reported that the concentrations of unchanged mepirapim in whole blood and urine were much higher than those of other synthetic cannabinoids. To determine the postmortem distribution of mepirapim and acetyl fentanyl in the deceased individual, we established a standard addition method for detailed analysis by liquid chromatography-mass spectrometry (LC-MS) for quantification of these drugs.

Cranio-morphometric and aDNA corroboration of the Austronesian dispersal model in ancient Island Southeast Asia: Support from Gua Harimau, Indonesia.

The Austronesian language is spread from Madagascar in the west, Island Southeast Asia (ISEA) in the east (e.g. the Philippines and Indonesian archipelagoes) and throughout the Pacific, as far east as Easter Island.

Identification and quantification of mepirapim and acetyl fentanyl in authentic human whole blood and urine samples by GC-MS/MS and LC-MS/MS.

Purpose: We encountered a curious case in which two male subjects self-administered mepirapim plus acetyl fentanyl by different routes, i.e., intravenously and by inhalation.

Ethnic derivation of the Ainu inferred from ancient mitochondrial DNA data.

Objectives: The Ainu, the indigenous people living on the northernmost island of Japan, Hokkaido, have long been a focus of anthropological interest because of their cultural, linguistic, and physical identity. A major problem with genetic studies on the Ainu is that the previously published data stemmed almost exclusively from only 51 modern-day individuals living in Biratori Town, central Hokkaido. To clarify the actual genetic characteristics of the Ainu, individuals who are less influenced by mainland Japanese, who started large-scale immigration into Hokkaido about 150 years ago, should be examined.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples.

The art of traditional native PAGE: The APLP 48-ID assay for human identification.

When full STR profiles cannot be obtained, further DNA analyses targeting single nucleotide polymorphisms (SNPs) may occasionally yield valuable information. Although the discrimination power of each SNP is relatively low, combined analysis of many SNPs can improve the personal identification ability to a level as high as that of commercial STR typing kits. In this study, we developed a new SNP typing method, named the amplified-product length polymorphism (APLP) 48-ID assay, for genotyping of 47 autosomal SNPs and two X and Y chromosomal markers for sex typing.

A Unique Primer with an Inosine Chain at the 5'-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method.

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3'-terminus of each primer. To use this method at least two allele-specific primers and one "counter-primer", which serves as a common forward or reverse primer of the allele-specific primers, are required.

Investigation of Japanese-specific alleles: most are of Jomon lineage.

Japanese-specific alleles are expected to be powerful markers for the differentiation of the Japanese from other people. In this study, three single nucleotide polymorphisms (SNPs) in the GALNT11, H19, and PLA2G12A genes were analyzed in 2396 DNA samples from 25 global populations, and the derived alleles suggested that Japanese-specific alleles exist on autosomes. To identify new Japanese-specific alleles, candidate SNPs obtained from the HapMap database were investigated using 875 DNA samples from nine populations.

A novel method for sex determination by detecting the number of X chromosomes.

A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data.

Comparison of Genomic and Epigenomic Expression in Monozygotic Twins Discordant for Rett Syndrome.

Monozygotic (identical) twins have been widely used in genetic studies to determine the relative contributions of heredity and the environment in human diseases. Discordance in disease manifestation between affected monozygotic twins has been attributed to either environmental factors or different patterns of X chromosome inactivation (XCI). However, recent studies have identified genetic and epigenetic differences between monozygotic twins, thereby challenging the accepted experimental model for distinguishing the effects of nature and nurture.

Identification of feces by detection of Bacteroides genes.

In forensic science, the identification of feces is very important in a variety of crime investigations. However, no sensitive and simple fecal identification method using molecular biological techniques has been reported. Here, we focused on the fecal bacteria, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron, and developed a novel fecal identification method by detection of the gene sequences specific to these bacteria in various body (feces, blood, saliva, semen, urine, vaginal fluids and skin surfaces) and forensic (anal adhesions) specimens.

A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification.

We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP).

[Evaluation of the sexing methods using the cranial traits in the Japanese population].

Nowadays, various morphological traits are routinely used for sexing the human skulls. The efficacy and reliability of sexing methods based on these traits in the Japanese population have not been systematically investigated. For sexing the skull, the authors established the well-defined criteria for sexing skulls by using the following five morphological traits; (1) the prominence and texture of the supraorbital arc; (2) the sharpness of the supraorbital margin; (3) the relative size of the zygomatic arc and the existence of a depression on it; (4) the size of the mastoid process and the existence of the supramastoid crest; (5) the prominence of the external occipital crest and the external occipital protuberance, and then evaluated their availability by using 313 recent Japanese skulls (205 males and 108 females) with known sex and age-at-death.

A novel method for the identification of saliva by detecting oral streptococci using PCR.

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva.