Improvement of C-to-U RNA editing using an artificial MS2-APOBEC system.

RNA cytidine deamination (C-to-U editing) has been achieved using the MS2-apolipoprotein B-editing catalytic polypeptide-like (APOBEC)1 editing system. Here, we fused the cytidine deaminase (CDA) enzymes APOBEC3A and APOBEC3G with the MS2 system and examined their RNA editing efficiencies in transfected HEK 293T cells. Given the single-stranded RNA preferences of APOBEC3A and APOBEC3G, we designed unconventional guide RNAs that induced a loop at the target sequence, allowing the target to form a single-stranded structure. Because APOBEC3A and APOBEC3G have different base preferences (5'-TC and 5'-CC, respectively), we introduced the D317W mutation into APOBEC3G to convert its base preference to that of APOBEC3A. Upon co-transfection with a guide RNA that induced the formation of a 14 nt loop on the target sequence, MS2-fused APOBEC3A and APOBEC3G showed high editing efficiency. While the D317W mutation of APOBEC3G led to a slight improvement in editing efficiency, the difference was not statistically significant. These findings indicate that APOBEC3A and APOBEC3G can induce C-to-U RNA editing when transfected with a loop guide RNA. Moreover, the editing efficiency of APOBEC3G can be enhanced by site-specific mutation to alter the base preference. Overall, our results demonstrate that the MS2 system can fuse and catalyze reactions with different enzymes, suggesting that it holds an even greater potential for RNA editing than is utilized currently.

Developmental validation of a complementary Y-STR system for the amplification of forensic samples.

In this study, a new complementary Y-STR system that includes 31 loci was developed (DYS522, DYS388, DYF387S1a/b, DYS510, DYS587, DYS645, DYS531, DYS593, DYS617, GATA_A10, DYS622, DYS552, DYS508, DYS447, DYS527a/b, DYS446, DYS459a/b, DYS444, DYS557, DYS443, DYS626, DYS630, DYS526a, DYF404S1a/b, DYS520, DYS518, and DYS526b). This 31-plex Y-STR system, SureID® Y-comp, is designed for biological samples from forensic casework and reference samples from forensic DNA database. To validate the suitability of this novel kit, many developmental works including size precision testing, sensitivity, male specificity testing, species specificity, PCR inhibitors, stutter precision, reproducibility, suitability for use on DNA mixture and parallel testing of different capillary electrophoresis devices were performed. Mutation rates were investigated using 295 DNA-confirmed father-son pairs. The results demonstrate that the SureID® Y-comp Kit is time-efficient, accurate, and reliable for various case-type samples. It possessed a higher discrimination power and can be a stand-alone kit for male identification. Moreover, the simply acquired additional Y-STR loci will be conductive to construct a robust database. Even if various commercial Y-STR kits are used in distinct forensic laboratories, a wider trans-database retrieval will become feasible with the effort of the SureID® Y-comp Kit.

Phylogenetic analyses of 41 Y-STRs and machine learning-based haplogroup prediction in the Qingdao Han population from Shandong province, Eastern China.

BACKGROUND: Known for its rich history and culture, Qingdao is a typical symbol of Chinese maritime culture. Its unique genetic landscape has aroused interest among geneticists and forensic scientists. However, the genetic landscape of Qingdao has never been uncovered. AIM: This investigation intends to provide light on Qingdao's paternal genetic diversity and its evolutionary connections to other Han subgroups. SUBJECTS AND METHODS: The genetic polymorphisms of 41 Y-chromosomal short tandem repeat (STR) loci in the Qingdao Han were investigated using SureID® PathFinder Plus Kit. Phylogenetic studies were performed using genotype data from 52 East Asian groups at 23 common Y-STR loci. A multidimensional scaling plot and cladogram were constructed. Linear Discriminant Analysis (LDA) was carried out for predicting categories among the Han people. The k-nearest neighbour (kNN) algorithm was utilised to designate Y-SNP haplogroups for each haplotype. RESULTS: The Qingdao Han were genetically far from the Tibeto-Burman populations and close with the Han people from northern China. LDA indicated a deep integration among the present-day Han people. By the kNN model, the predicted O2a2 and O2a1 were shown to be the predominant Y-SNP haplogroups. CONCLUSIONS: This study would be helpful for reconstructing the patrilineal history in China and establishing a more comprehensive Y-STR database.

The Role of the Exonic lncRNA PRKDC-210 in Transcription Regulation.

In recent years, long noncoding RNAs (lncRNAs) have received increasing attention and have been reported to be associated with various genetic abnormalities. However, the functions of many lncRNAs, including those of long exonic noncoding RNAs (lencRNAs), have not yet been elucidated. Here, we used a novel tethering luciferase assay to analyze the transcriptional regulatory functions of five lencRNAs that are upregulated in cancer. We found that the lencRNA PRKDC-210 interacts with MED12, a component of the CDK8 complex, to regulate the transcription of several genes. The transcriptional activation ability of PRKDC-210 was abolished in siRNA-treated CDK8-depleted cells. We also confirmed the enrichment of PRKDC-210 on RNA polymerase II. RNA-seq analysis of cells in which PRKDC-210 or PRKDC mRNA was knocked down using antisense oligonucleotides revealed that PRKDC-210 can affect the expression levels of genes related to fatty acid metabolism. Finally, we used a ChIRP assay to examine PRKDC-210-enriched sites in the genome. Overall, our findings demonstrate that the lencRNA PRKDC-210 promotes transcription through the CDK8 complex pathway at the transcription initiation site. We propose that PRKDC-210 can affect the transcription of adjacent genes after its transcription and splicing.

Assessing the factors influencing the performance of machine learning for classifying haplogroups from Y-STR haplotypes.

Two distinct genetic markers, single nucleotide polymorphisms (Y-SNPs) and short tandem repeats (Y-STRs), exist simultaneously in the non-recombining portion of the Y chromosome. Because of their different rates of mutation, Y-STRs and Y-SNPs play distinct roles in forensic and evolutionary genetics. Current approaches to infer haplogroup status rely on genotyping lots of Y-SNP loci. Given the relationship between haplotype and haplogroup of a Y chromosome, a cost-effective strategy of Y-STRs typing had an advantage in haplogroup prediction. Many machine learning algorithms have sprung up for assigning a Y-STR haplotype to a haplogroup. However, a series of issues must be solved before the using of machine learning method in practice. Thus, the k-nearest neighbor (kNN) classifier was built respectively based on different situations in this study. We assessed different factors which may influence the performance of the kNN prediction model for classifying haplogroups. The training set was based on a diverse ground-truth data set comprising Y-STR haplotypes and corresponding Y-SNP haplogroups. Our results showed that combining different levels of haplogroups into the observations or transracial prediction was impractical. Moreover, using more slow mutation Y-STR loci in the category is good for promoting classification accuracy. The preconditions for an effective and accurate haplogroup assignment by the kNN classifier were revealed.

Phylogenetic analyses of the 19 STR loci in X chromosome revealed discriminant forensic characteristics for Chinese Daur and Oroqen minorities.

In respect of forensic genetics, X-STRs are widely applied for deficiency paternity cases. Given the popularization of AGCU-X19 STR Kit in China, there has been investigation conducted into the genetic data and forensic parameters of 19 X-STR loci in many of the Chinese ethnic groups, which makes it possible to perform nationwide phylogenetic comparation. To evaluate the allele and haplotype diversity of 19 X-STR loci and to explore their forensic efficiency in the Daur and Oroqen minorities, unrelated healthy Daur (n = 86) and Oroqen (n = 165) individuals were recruited from Heilongjiang province, so as to reveal the phylogenetic relationship between the two minorities and other Chinese ethnic groups. Of the Daur and Oroqen minorities, 172 and 183 alleles at the 19 X-STR loci were observed, respectively. Haplotype diversity exceeded 0.9 among all the linkage clusters. High cumulative value was observed for the power of discrimination, the probability of exclusion, and the mean exclusion chance for deficiency cases (normal trios and duo cases). As revealed by this study, the panel of 19 X-STR loci is an effective supplementary tool for the kinship test of the studied nationalities.

Developmental validation study of a 32-plex STR direct amplification system for forensic reference samples.

The STRtyper-32G PCR Amplification Kit is a 6-dye multiplex system that combines the 30 autosomal STR loci with an Indel site (YIndel) and the sex-determinant locus Amelogenin. In addition to more loci, Master Mix has been optimized to amplify DNA on different substrates. The autosomal STR loci contained in this novel system meet the compatibility of requirements for databasing. In this study, the developmental validation study of the STRtyper-32G Kit followed the guidelines of SWGDAM (Scientific Working Group on DNA Analysis Methods), including PCR-based studies, species specificity, inhibitors, sensitivity, precision, repeatability, stutter, DNA mixtures, concordance studies, and population genetics studies. The validation results indicate that the new multiplex system is a robust tool for forensic database applications.

Population genetic characteristics for populations on the Shandong Peninsula revealed by autosomal STR.

The Shandong Peninsula is the largest peninsula in China and has played a vital part in Chinese civilisation. The ancient independent Laizi kingdom was located on the Shandong peninsula. While large demographic changes have happened at this peninsula throughout history, the genetic landscape of modern populations on this peninsula has never been clarified. The aims of our study were to investigate population genetic characteristics of the populations on the Shandong peninsula and to reveal their genetic affinities with other populations around the world. Allele frequencies, Hardy-Weinberg equilibrium, and forensic parameters of 15 autosomal STRs in the AmpFlSTR® Identifiler system were obtained from the studied populations with 2441 individuals in total. Allele frequencies were used to reveal the phylogenetic relationships among 287 worldwide populations. The combined power of discrimination (CPD) and the combined power of exclusion (CPE) in the whole of the Han population on the Shandong Peninsula were 0.999999999999999983 and 0.999998155, respectively. The 15 autosomal loci were polymorphic and informative among our studied populations. Genetic homogeneities were revealed between the modern populations on the Shandong Peninsula and Han nationalities from Northeastern China as well as East China.

Technical note: developmental validation of a novel 41-plex Y-STR system for the direct amplification of reference samples.

The SureID® PathFinder Plus is a new 6-dye, 41-plex Y-STR system that includes the 17 loci from the Yfiler® kit (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) plus 14 rapidly mutating Y-STR loci (DYS449, DYS481, DYS518, DYS527a/b, DYS533, DYS549, DYS570, DYS576, DYS627, DYF387S1a/b, and DYF404S1), and 10 low-medium mutation loci (DYS388, DYS444, DYS447, DYS460, DYS522, DYS557, DYS593, DYS596, DYS643, and DYS645). The inclusion of the 14 rapidly mutating Y-STR loci improves the discrimination of related individuals. Conversely, the 10 low-medium mutation loci are suitable not only for familial searching but also for providing a higher refinement in the construction of Y chromosome phylogenetic relationships among lineages. The 41-plex Y-STR system is designed for direct amplification of reference samples, such as blood samples on an FTA® Card, gauze, tissue, or cotton substrates as well as hair root or buccal samples on swabs. We performed developmental validation work including accuracy, stability, stutter precision, species specificity, sensitivity, PCR inhibitors, reproducibility, parallel testing of the system, and suitability for use on DNA mixtures. In addition, mutations of the loci were analyzed by 754 DNA-confirmed father-son pairs. The results demonstrate that this kit, developed in-house, is time-efficient, accurate, reliable, and highly informative for forensic database, familial searching, and distinguishing related males.

Gene flow and phylogenetic analyses of paternal lineages in the Yi-Luo valley using Y-STR genetic markers.

BACKGROUND: The Yi-Luo valley witnessed the most significant socio-political transformation of China and was deeply implicated in several enormous migrations of the Han population. However, little has been done to clarify its paternal genetic variation or phylogenetic relationship, particularly concerning the genetic evidence of their migrations. AIM: This study aims to uncover the population genetic characteristics in the Yi-Luo valley and provide genetic evidence for its people's migrations. SUBJECTS AND METHODS: Seventeen Y-STR loci included in the AmpFlSTR®Yfiler™ were typed in 2,314 individuals from seven different regions along the Yi-Luo valley. A multidimensional scaling plot and neighbor-joining tree were constructed for nationwide genetic comparisons. Y-haplogroup frequencies and migration rates were estimated among the studied populations. Gene flows were detected by different migration models and directions. RESULTS: The predicted Y-haplogroups demonstrated the predominance of O2a2. Genetic affinities were observed among Han, Hakka, Danmin, and Bai. Anhui was shown to be the most crucial transfer spot for the Hakkas when they moved out of the Central Plains to South China. CONCLUSIONS: This study reveals the genetic landscape of paternal lineages living in the Yi-Luo valley and enriches our understanding of the great migration in Chinese history.

Forensic and phylogenetic analyses of populations in the Tibetan-Yi corridor using 41 Y-STRs.

Y-chromosome haplotypes of 527 non-related males (176 Han, 186 Tibetan, and 165 Yi) in the Tibetan-Yi corridor were analyzed using SureID® PathFinder Plus. In the populations of Han, Tibetans, and Yi, the haplotype diversity was 0.9989, 0.9981, and 0.9993, respectively, and the discrimination capacity was 0.9148, 0.8925, and 0.9576, respectively. Phylogenetic relationships among 12 studied ethnic groups and 7 other ethnic groups in the Tibetan-Yi corridor were investigated. Both multi-dimensional scaling analysis and phylogenetic reconstructions indicated that Tibetans appeared separated from the Han and Yi ethnic groups in the Tibetan-Yi corridor. Their genetic homogeneity or heterogeneity has not entirely been affected by their geographical distance and linguistic origin.

Phylogenic analysis and forensic genetic characterization of Guizhou Miao tribes from 58 microareas via autosomal STR.

Genetic polymorphism of 17 autosomal short tandem repeat (STR) loci, included in the PowerPlex®18D amplification kit, were analyzed in Miao tribes from 58 different sampling microareas (N = 5255) of Guizhou as well as two cities (N = 151) of Hunan, China. Allele frequencies and forensic efficiency parameters were calculated. Moreover, comprehensive population genetic comparisons among 91 nationwide populations and 174 Asian populations were conducted based on raw genotype data and allele frequency data, respectively. Our results of forensic efficiency parameters showed that the panel was a robust tool in forensic individual identification and paternity cases for this population. Genetic affinities were observed among most of the Miao tribes revealed by multidimensional scaling plot, principal component analysis, and neighboring-joining tree. The genetic distance between Miao tribes and Han nationalities were varies by different geographical positions. Some of the Miao tribes were genetically closer to the Hmong-Mien populations living in southeastern contiguous regions and even the Indochina. The result coincided with the migration or reverse migration routes for Miao nationality in modern history. This study of the Miao tribes from plenty of microareas in Guizhou would be useful in reconstructing the population history and establishing a more comprehensive forensic reference database.

Forensic and phylogenetic analyses among three Yi populations in Southwest China with 27 Y chromosomal STR loci.

We have analyzed the Y chromosome haplotypes of 510 non-related Yi males grouped in three Chinese provinces (Guizhou, Sichuan, and Yunnan) using the Yfiler® Plus Kit. A total of 484 haplotypes were detected, out of which 460 were unique. The observed haplotype diversity and discrimination capacity were 0.9998 and 0.9490, respectively. To investigate the genetic relationship of the studied population, multidimensional scaling (MDS) plot and neighbor-joining (NJ) tree phylogenetic analyses were constructed which demonstrated that the Y chromosome lineage among the Yi ethnicity was different.

Haplotype diversity of 17 Y-chromosome STR loci in Han population from different areas of Sichuan Province, Southwest China.

The distribution of 17 Y-chromosome short tandem repeat (STR) loci, included in the AmpFlSTR®Yfiler™ amplification kit, were analyzed in six different samplings (N=878) from Sichuan, China. Haplotype diversity and discrimination capacity (DC) values were calculated. Pairwise Rst values were evaluated in AMOVA analysis and visualized through multidimensional scaling (MDS). A total of 547 unique haplotypes were detected. The observed haplotype diversity and discrimination capacity were 0.9995 and 0.7745, respectively. The homogeneity of Sichuan Han population was detected when microareas were analyzed. This population exhibited no significant genetic difference to both of the minorities in reference databases, Mongolian and Manchu, which had been through mass ethnic amalgamation with Sichuan Han population in history.

Revisiting the male genetic landscape of China: a multi-center study of almost 38,000 Y-STR haplotypes.

China has repeatedly been the subject of genetic studies to elucidate its prehistoric and historic demography. While some studies reported a genetic distinction between Northern and Southern Han Chinese, others showed a more clinal picture of small differences within China. Here, we investigated the distribution of Y chromosome variation along administrative as well as ethnic divisions in the mainland territory of the People's Republic of China, including 28 administrative regions and 19 recognized Chinese nationalities, to assess the impact of recent demographic processes. To this end, we analyzed 37,994 Y chromosomal 17-marker haplotype profiles from the YHRD database with respect to forensic diversity measures and genetic distance between groups defined by administrative boundaries and ethnic origin. We observed high diversity throughout all Chinese provinces and ethnicities. Some ethnicities, including most prominently Kazakhs and Tibetans, showed significant genetic differentiation from the Han and other groups. However, differences between provinces were, except for those located on the Tibetan plateau, less pronounced. This discrepancy is explicable by the sizeable presence of Han speakers, who showed high genetic homogeneity all across China, in nearly all studied provinces. Furthermore, we observed a continuous genetic North-South gradient in the Han, confirming previous reports of a clinal distribution of Y chromosome variation and being in notable concordance with the previously observed spatial distribution of autosomal variation. Our findings shed light on the demographic changes in China accrued by a fast-growing and increasingly mobile population.

Use of multi-InDels as novel markers to analyze 13 X-chromosome haplotype loci for forensic purposes.

Many studies have been proposed to identify insertion/deletion (InDel) polymorphisms in humans for forensic genetic studies. However, the discriminatory power of InDels is limited by the poor polymorphisms of diallelic markers. To improve their discriminatory power, we developed multi-InDel, a novel autosomal marker comprising more than two InDel loci that are tightly linked by their physical position and combined into a specific marker by a pair of PCR primers. This strategy gives at least three haplotypes for each multi-InDel marker. Such markers can be potentially very useful in forensic applications. In this study, we focused on multi-InDel markers located on X chromosome (ChrX). A multiplex system with 13 multi-InDel markers, including 28 InDel loci in ChrX, was developed. To validate the multi-InDel panel, the haplotype distribution in a population sample and in a set of pedigrees was investigated. This study demonstrates usefulness of these markers for individual identification and relationship studies. We highlight the fact that the multi-InDel markers located on ChrX can provide new supporting information for complex kinship testing.

Population genetics for 23 Y-STR loci in Tibetan in China and confirmation of DYS448 null allele.

Tibetan is one of 56 ethnic groups in China, where a level of genetic sub-structure might be expected. Although a global analysis of Y-chromosomal haplotype diversity for 23 STR loci and Y-STR databases with PPY23 kit were created with collaborative effort, there was a lack of data for Tibetan population. In this study we evaluated 248 unrelated male individuals of Chinese Tibetan living in the Tibet Autonomous Region to explore the underlying genetic structure of Tibetan populations. These samples were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) by using PPY23 kit. A total of 224 different haplotypes were found. Haplotype diversity was 0.9990. Both Rst pairwise analyses and multidimensional scaling plot showed the genetic structure of Tibetan population was significantly different from some of Chinese ethnic groups and neighboring populations. There were few interesting null features at DYS448 observed by PPY23 that deserved some comment. It revealed that PPY23 marker set provided substantially stronger discriminatory power in Tibetan population.

[Advances in identification of semen stains].

Stain identification has long been a task in forensic biology. The identification of semen stain, one of the most common human stains, can provide crucial information for crime scene reconstruction and forensic investigation. Traditional detection of semen stain depends largely on the microscopic identification of spermatozoa, enzyme activity-based methods or antigen-antibody reactions. These morphological, proteinological and zymological approaches, however, are apparently inadequate in identifying tiny, admixed, degraded or contaminated samples. With the development of transcriptomics and epigenetics, many semen-specific mRNA markers, such as protamine-1 (PRM1) and -2 (PRM2), have been applied to semen and semen stain identification. Messenger RNA profiling shows great promise in identifying tissues as demonstrated by the recognition of specific markers. Further more, studies on tis-sue-specific differential DNA methylation will provide a scrumptious way of identifying difficult samples.