Lagabaiyila Zha

Potential use of bacterial community succession for estimating post-mortem interval as revealed by high-throughput sequencing

Decomposition is a complex process involving the interaction of both biotic and abiotic factors. Microbes play a critical role in the process of carrion decomposition. In this study, we analysed bacterial communities from live rats and rat remains decomposed under natural conditions, or excluding sarcosaphagous insect interference, in China using Illumina MiSeq sequencing of 16S rRNA gene amplicons. A total of 1,394,842 high-quality sequences and 1,938 singleton operational taxonomic units were obtained. Bacterial communities showed notable variation in relative abundance and became more similar to each other across body sites during the decomposition process. As decomposition progressed, Proteobacteria (mostly Gammaproteobacteria) became the predominant phylum in both the buccal cavity and rectum, while Firmicutes and Bacteroidetes in the mouth and rectum, respectively, gradually decreased. In particular, the arrival and oviposition of sarcosaphagous insects had no obvious influence on bacterial taxa composition, but accelerated the loss of biomass. In contrast to the rectum, the microbial community structure in the buccal cavity of live rats differed considerably from that of rats immediately after death. Although this research indicates that bacterial communities can be used as a “microbial clock” for the estimation of post-mortem interval, further work is required to better understand this concept.

Genetic polymorphism of 21 non-CODIS STR loci in the Chinese Mongolian ethnic minority.

In this research, we investigated the allele frequencies and forensic parameters of 21 non-, CODIS short tandem repeat (STR) loci (D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D1S1627, D3S4529, D2S441, D6S1017, D4S2408, D19S433, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D9S1122, D2S1776, D10S1435 and D5S2500) among 523 unrelated, Chinese Mongolians in the city of Tongliao, Horqin district, Inner Mongolia Autonomous Region.

Parallel Analysis of 124 Universal SNPs for Human Identification by Targeted Semiconductor Sequencing

SNPs, abundant in human genome with lower mutation rate, are attractive to genetic application like forensic, anthropological and evolutionary studies. Universal SNPs showing little allelic frequency variation among populations while remaining highly informative for human identification were obtained from previous studies. However, genotyping tools target only dozens of markers simultaneously, limiting their applications. Here, 124 SNPs were simultaneous tested using Ampliseq technology with Ion Torrent PGM platform. Concordance study was performed with 2 reference samples of 9947A and 9948 between NGS and Sanger sequencing. Full concordance were obtained except genotype of rs576261 with 9947A. Parameter of FMAR (%) was introduced for NGS data analysis for the first time, evaluating allelic performance, sensitivity testing and mixture testing. FMAR values for accurate heterozygotes should be range from 50% to 60%, for homozygotes or Y-SNP should be above 90%. SNPs of rs7520386, rs4530059, rs214955, rs1523537, rs2342747, rs576261 and rs12997453 were recognized as poorly performing loci, either with allelic imbalance or with lower coverage. Sensitivity testing demonstrated that with DNA range from 10 ng-0.5 ng, all correct genotypes were obtained. For mixture testing, a clear linear correlation (R2 = 0.9429) between the excepted FMAR and observed FMAR values of mixtures was observed.

The complete mitochondrial genome of the flesh fly, Muscina stabulans (Diptera: muscidae)

Abstract In this study, we sequence the complete mitochondrial genome of Muscina stabulans (Diptera: Muscidae), a forensically important entomology, for the first time. The 15,933 bp circular genome contains the 37 genes found in a typical metazoan genome: 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 non-coding A + T-rich region. All the protein initiation codons are ATN, except for cox1 and ND1 that begin with TCG and TTG. The arrangement of the genes was the same as that found in the other insect. The overall base composition on heavy strand was as follows A: 40.00%, G: 8.80%, C: 13.34%, T: 37.86% and the A + T content 77.86%. The mitochondrial genome of Muscina stabulans presented will be valuable for resolving phylogenetic relationships within the family Muscidae and order Diptera, and could be used to identify favourable genetic markers for species identifications for forensic purposes.

Genetic polymorphic investigation of 21 autosomal short tandem repeat loci in the Chinese Li ethnic group

Abstract Population genetic data of 21 autosomal short tandem repeats (STRs) were obtained in a sample of 106 unrelated healthy individuals of Bai ethnic minority born in the Dali Bai Autonomous Prefecture in Yunnan Province. We observed 138 alleles with corresponding allelic frequencies ranging from 0.005 to 0.575. The genotypic frequency distributions at those STR loci were consistent with Hardy–Weinberg equilibrium (Bonferronis correction was used for Hardy–Weinberg equilibrium tests). The combined probability of exclusion, power of discrimination, probability of matching value for all 21 STR loci were 0.9999975729, 0.999999999999999999872 and 1.28×10 −19 , respectively. The population data in this study showed significant differences from the previously published population data of Tibetan and Salar groups in some loci.

Population genetic study of 34 X-Chromosome markers in 5 main ethnic groups of China

As a multi-ethnic country, China has some indigenous population groups which vary in culture and social customs, perhaps as a result of geographic isolation and different traditions. However, upon close interactions and intermarriage, admixture of different gene pools among these ethnic groups may occur. In order to gain more insight on the genetic background of X-Chromosome from these ethnic groups, a set of X-markers (18 X-STRs and 16 X-Indels) was genotyped in 5 main ethnic groups of China (HAN, HUI, Uygur, Mongolian, Tibetan). Twenty-three private alleles were detected in HAN, Uygur, Tibetan and Mongolian. Significant differences (p < 0.0001) were all observed for the 3 parameters of heterozygosity (Ho, He and UHe) among the 5 ethnic groups. Highest values of Nei genetic distance were always observed at HUI-Uygur pairwise when analyzed with X-STRs or X-Indels separately and combined. Phylogenetic tree and PCA analyses revealed a clear pattern of population differentiation of HUI and Uygur. However, the HAN, Tibetan and Mongolian ethnic groups were closely clustered. Eighteen X-Indels exhibited in general congruent phylogenetic signal and similar cluster among the 5 ethnic groups compared with 16 X-STRs. Aforementioned results proved the genetic polymorphism and potential of the 34 X-markers in the 5 ethnic groups.

Comparative Mitogenomic Analysis of Forensically Important Sarcophagid Flies (Diptera: Sarcophagidae) and Implications of Species Identification

Abstract The flesh flies (Diptera: Sarcophagidae) are significant in forensic investigations. The mitochondrial genome (mitogeome) has been widely used as genetic markers for phylogenetic analysis and species identification. To further understand the mitogenome-level features in Sarcophagidae, the complete mitogenome of Sarcophaga formosensis (Kirneret Lopes, 1961) (Diptera: Sarcophagidae) and Sarcophaga misera (Walker, 1849) (Diptera: Sarcophagidae) was firstly sequenced, annotated, and compared with other 13 Sarcophagidae species. The result indicated that the gene arrangement, gene content, base composition, and codon usage were conserved in the ancestral arthropod. Evolutionary rate of the mitogenome fragments revealed that the nonsynonymous and synonymous substitution rates (Ka and Ks) ratio was less than 1.00, indicating these variable sites under strong purifying selection. Almost all transfer RNA genes (tRNAs) have typical clover-leaf structures within these sarcophagid mitogenomes, except tRNA-Ser (AGN) is lack of the dihydrouridine arm. This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the Sarcophagidae. Phylogenetic analyses containing the interspecific distances from different regions in these species provided us new insights into the application of these effective genetic markers for species identification of flesh flies.

The Application of COI Gene for Species Identification of Forensically Important Muscid Flies (Diptera: Muscidae)

Abstract Muscid Flies (Diptera: Muscidae) are of great forensic importance due to their wide distribution, ubiquitous and synanthropic nature. They are frequently neglected as they tend to arrive at the corpses later than the flesh flies and blow flies. Moreover, the lack of species-level identification also hinders investigation of medicolegal purposes. To overcome the difficulty of morphological identification, molecular method has gained relevance. Cytochrome c oxidase subunit I (COI) gene has been widely utilized. Nonetheless, to achieve correct identification of an unknown sample, it is important to survey certain muscid taxa from its geographic distribution range. Accordingly, the aim of this study is to contribute more geographically specific. We sequenced the COI gene of 51 muscid specimens of 12 species, and added all correct sequences available in GenBank to yield a total data set of 125 COI sequences from 33 muscid species to evaluate the COI gene as a molecular diagnostic tool.The interspecific distances were extremely high (4.7–19.8%) in either the standard barcoding fragment (658 bp) or the long COI sequence (1,019–1,535 bp), demonstrating that these two genetic markers were nearly identical in the species identification. However, the intraspecific distances of the long COI sequences were significantly higher than the barcoding region for the conspecific species that geographical locations vary greatly.Therefore, genetic diversity presented in this study provides a reference for species identification of muscid flies. Nevertheless, further investigation and data from more muscid species are required to enhance the efficacy of species-level identification using COI gene as a genetic marker.

The forensic value of X-linked markers in mixed-male DNA analysis

Autosomal genetic markers and Y chromosome markers have been widely applied in analysis of mixed stains at crime scenes by forensic scientists. However, true genotype combinations are often difficult to distinguish using autosomal markers when similar amounts of DNA are contributed by multiple donors. In addition, specific individuals cannot be determined by Y chromosomal markers because male relatives share the same Y chromosome. X-linked markers, possessing characteristics somewhere intermediate between autosomes and the Y chromosome, are less universally applied in criminal casework. In this paper, X markers are proposed to apply to male mixtures because their true genes can be more easily and accurately recognized than the decision of the genotypes of AS markers. In this study, an actual two-man mixed stain from a forensic case file and simulated male-mixed DNA were examined simultaneously with the X markers and autosomal markers. Finally, the actual mixture was separated successfully by the X markers, although it was unresolved by AS-STRs, and the separation ratio of the simulated mixture was much higher using Chr X tools than with AS methods. We believe X-linked markers provide significant advantages in individual discrimination of male mixtures that should be further applied to forensic work.

Developing a MtSNP-based genotyping system for genetic identification of forensically important flesh flies (Diptera: Sarcophagidae)

Some representatives of flesh flies visiting/colonizing the decomposed remains demonstrated their values in estimating the minimal postmortem interval (PMImin) since death. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. Although mitochondrial genes have been proposed as a potential DNA barcode for the species-level identification of sarcophagids, some defects still remain such as the substantial memory and processing time taken for homologous comparisons online. Moreover, species identification is mainly achieved by Sanger sequencing based on PCR with genus-specific primers. In the present study we characterized 24 single nucleotide polymorphisms (SNPs) as robust markers of genetic variation for identifying different sarcophagids based on available cytochrome c oxidase I (COI) data and verified them through pyrosequencing (PSQ) technology to establish a SNP-based genotyping system. The system provides a preliminary foundation for developing a rapid, reliable, and high-throughput assay so as to efficiently and accurately identify the sarcophgid flies. Furthermore, the PSQ approach is proved to be faster, more cost-effective as well as more sensitive and specific than custom Sanger sequencing.