A novel multiplex of 12 multicopy Y-STRs for forensic application.

Y chromosomal short tandem repeats (Y-STRs) have been applied overwhelmingly in forensic areas for solving paternity identification and sexual assault cases. Yet the widely used Y-STR kits contain mostly single-copy markers, which may restrict the discrimination power. Here, a novel Y-STR multiplex was developed and validated in order to complement the currently available Y-STR kits, especially on differentiating male relatives. The assay includes twelve multicopy Y-STR loci (DYF371, DYF383S1, DYS385, DYF387S1, DYS389I/II, DYF399S1, DYF404S1, DYF409S1, DYF411S1, DYS464, DYS526, DYS527; four of them are rapidly mutating ones), 1 single-copy Y-STR (DYS391), and Amelogenin, and was optimized to amplify at annealing temperature of 59°C and 28 cycles. Validation studies show that full profiles are obtained with 0.125 ng of male DNA. The system is capable of overcoming high concentrations of inhibitors such as hematin, EDTA, and humic acid. Besides, the results demonstrate good sizing precision and the ability to detect male-specific profiles in male/female DNA mixtures at a ratio of 1:800. Excellent species specificity was also observed in microorganisms and non-primates, while detectable peaks were found in some primates. Based on published genetic data, gene diversity values were above 0.7 for most of the loci in our multiplex, inferring a high capacity in discriminating unrelated and related male individuals. The kit is of great potential for forensic application.

Genome and transcriptome of Papaver somniferum Chinese landrace CHM indicates that massive genome expansion contributes to high benzylisoquinoline alkaloid biosynthesis.

Opium poppy (Papaver somniferum) is a source of morphine, codeine, and semisynthetic derivatives, including oxycodone and naltrexone. Here, we report the de novo assembly and genomic analysis of P. somniferum traditional landrace 'Chinese Herbal Medicine'. Variations between the 2.62 Gb CHM genome and that of the previously sequenced high noscapine 1 (HN1) variety were also explored. Among 79,668 protein-coding genes, we functionally annotated 88.9%, compared to 68.8% reported in the HN1 genome. Gene family and 4DTv comparative analyses with three other Papaveraceae species revealed that opium poppy underwent two whole-genome duplication (WGD) events. The first of these, in ancestral Ranunculales, expanded gene families related to characteristic secondary metabolite production and disease resistance. The more recent species-specific WGD mediated by transposable elements resulted in massive genome expansion. Genes carrying structural variations and large-effect variants associated with agronomically different phenotypes between CHM and HN1 that were identified through our transcriptomic comparison of multiple organs and developmental stages can enable the development of new varieties. These genomic and transcriptomic analyses will provide a valuable resource that informs future basic and agricultural studies of the opium poppy.

Development and validation of a new 18 X-STR typing assay for forensic applications.

With a unique inheritance pattern compared to autosomal short tandem repeats (A-STRs), X chromosomal STRs (X-STRs) have special usage in forensic relationship testing. In this study, we designed a multiplex amplification system (named TYPER-X19 multiplex assay) consisting of 18 STR loci spreading from 7.837 to 149.460 Mb on the X chromosomes (DXS9895, DXS8378, DXS9902, DXS6810, DXS7132, DXS10079, DXS6789, DXS7424, DXS101, DXS6797, DXS7133, DXS6804, GATA165B12, DXS10103, HPRTB, GATA31E08, DXS8377, and DXS7423), and the amelogenin. PCR primers were marked with four kinds of fluorophores including FAM, HEX, TAMRA, and ROX. The multiplex system was optimized and tested for precision, concordance, reproducibility, sensitivity, stability, DNA mixture, and species specificity according to the conventional validation guidelines. The results indicated that the system was accurate, reliable, and sensitive enough, and was suitable for common forensic case-type samples. In the population genetic study, a total of 148 alleles were detected at the 18 X-STR loci in 398 Southern Han Chinese. Relatively high combined power of discrimination in male (PDm ), power of discrimination in female (PDf ), mean paternity exclusion chance in trios (MECtrio ), and mean paternity exclusion chance in duos (MECDuo ) by Desmarais were detected, and HPRTB-DXS10103 was in linkage disequilibrium. The results suggested that the TYPER-X19 multiplex assay was suitable for forensic applications.

Comparative evaluation of autosomal STRs and X-chromosome STRs as a complement of autosomal STRs in kinship testing in Southern Han Chinese.

Nowadays, kinship testing is very common in forensic caseworks, but the power of autosomal short tandem repeats (A-STRs) may be limited in complex cases. X-Chromosome short tandem repeats (X-STRs), having a unique heritage mode, should be of special use in some deficient cases. To evaluate and compare the potential of A-STR and X-STR as supplement genetic markers in deficient kinship testing, we simulated 10,000 duos for each of 18 kinds of relationships involving full sibling, half-sibling, grandparent-grandchild, and uncle/aunt-nephew/niece. Loci from STRTyper10, PowerPlex 16, and Investigator Argus X-12 were studied in Southern Han Chinese and the distribution of likelihood ratio (LR) values was analysed. With the addition of the X-12 system, the distribution of LR values for the full sisters, paternal half-sisters, paternal grandmother-granddaughters, maternal aunt-nieces, and maternal aunt-nephews separated much more obviously from those of unrelated duos, and the effectiveness was 1.0000, 0.99865, 0.9991, 0.8996 and 0.9634, respectively, which was more efficient than A-STRs. For the individual duos with other relationships, the effects of adding X-STRs and A-STRs were similar. Therefore, for the Southern Han Chinese, X-STRs can be very useful in kinship testing involving full sisters, paternal half-sisters, paternal grandmother-granddaughters, and maternal aunt-nieces/nephews.

Population genetic data of 4 multicopy Y-STR markers in Chinese.

Novel Y chromosomal STR (Y-STR) markers have been continuously discovered during the past decades, promoting the widely application of Y-STRs in the area of forensic science. Here, four multicopy Y-STR markers were focused, including DYF383S1, DYF409S1, DYF411S1 and DYF371, which are rarely reported in China and differ in the number of copies on Y chromosome. Characterization of the markers was performed in population of Hunan province, China, based on sequence analysis. Allele nomenclature and allelic ladder were then developed to avoid the disunity of typing standard. To evaluate their forensic performance, gene diversity of the four loci was investigated in 548 unrelated male individuals from Hunan population. The number of haplotype was analyzed by both conservative (C-type) and expanded approach (E-type) for markers containing more than 2 copies. As detected, there were 7, 9, 13 alleles and 15, 22, 23 haplotypes for DYF383S1, DYF409S1 and DYF411S1, respectively. Thirty-two C-types and 56 E-types were found in DYF371, indicating the highest haplotype diversity (HD) among all tested loci (0.871 and 0.888 for C-type and E-type, respectively). Two other Y-STRs (DYF409S1, DYF411S1) also showed high haplotype diversity (>0.8) in the population. Combining the four loci, discrimination capacity reached 0.505 (C-type) or 0.533 (E-type), and the total HD values exceeded 0.991. The results inferred great potential of the multicopy markers to improve the resolution of paternal identification in China population.

Development of a new 25plex STRs typing system for forensic application.

We have developed a novel STR 25-plex florescence multiplex-STR kit (DNATyper25) to genotype 23 autosomal and two sex-linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non-CODIS. The sex-linked markers include a Y-STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non-human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/µL and hematin concentration <400 µM. For forensic evaluation, the 23 autosomal STRs followed the Hardy-Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10-22 , TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.

Genetic diversities and phylogenetic analysis of 18 autosomal STR loci in the Uyghur population living in Ili, Northwest China.

Ili is located in northernmost Xinjiang, China. The Uyghur population only accounts for 15.90% of the total population in the nation. There is currently no large population data-based data set in Ili Uyghur. In this study, we investigated the genetic diversities of 18 autosomal short tandem repeat (STR) loci in 1129 Uyghur individuals living in Ili. The values of combined power of discrimination (CPD) and combined probability of exclusion (CPE) were 0.99999999999999999999990244 and 0.99999995645, respectively. Furthermore, we explored the genetic relationships between the Ili Uyghur population and 32 previously published populations. The results indicated that the Ili Uyghur population was more closely related to the Xinjiang Kazakh population. In addition, It was worth noting that significant differences were observed between Ili the Uyghur population and the Uyghur1 and Uyghur2 populations at the shared 15 loci, with significant differences at 7 and 11 loci after Bonferroni adjustment (p = 0.05/495 ≈ 0.00010).

Developmental validation of the DNATyper™Y26 PCR amplification kit: An enhanced Y-STR multiplex for familial searching.

The DNATyper™Y26 PCR Amplification kit, which including 26 low-medium mutating Y-STRs, is designed for Y-STR familial searching casework. The kit combines nine new Y-STR loci in addition to the 17 Y-STR loci from the commercially available AmpFlSTR®Yfiler® kit. The validation of the DNATyper™Y26 kit was performed in terms of technical index, including accuracy, stability, species specificity, sensitivity, adaptability for various samples, and mixture. Further, mutations of the 26 Y-STRs were analyzed by 1167 DNA-confirmed father-son pairs, and the results indicated that these loci had low or medium mutation rates. Furthermore, these Y-STRs loci were also tested in 1072 random male samples from Henan, Shanxi, Inner Mongolia, and Chongqing in China, showing their high power for forensic discrimination in the Chinese population. Thus, the DNATyper™Y26 PCR Amplification kit is a powerful tool for 'Y-STRs familial searching' in actual sexual-assault cases, indicating its unique advantage in familial searching due to Y-STR loci with only low-medium mutation rates.

Genetic analysis of 29 Y-STR loci in Han population from Dongfang, Southern China.

In the present study, blood samples of 984 unrelated Han individuals were collected from Dongfang, Southern China, after informed consent. A total of 29 Y-chromosomal short tandem repeat (Y-STR) were analyzed, including DYF387S1, DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS444, DYS447, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS508, DYS518, DYS533, DYS576, DYS635, DYS643 and GATAH4. A total of 749 different haplotypes were found among 984 individuals, of which 645 were unique. The haplotype diversity was 0.9988 and the discrimination capacity was 0.7612, while the match probability was 0.0025. The smallest genetic distance (RST = 0.0155) was found between the Dongfang Han population and Guizhou Han population, while the largest genetic distance (RST = 0.1284) was observed with Gansu Tibetan.

Concordance of mitochondrial DNA sequencing methods on bloodstains using Ion PGM™.

In this study, the complete mitochondrial genome (mtGenome) of six samples from three forensic cases was sequenced using the Ion Torrent Personal Genome Machine (PGM). The analyzed samples from forensic cases included bloodstains from several materials, such as gauze, Flinder's Technology Associates (FTA) cards and swabs. The age of the samples ranged from two months to twelve years. The complete mtGenomes were amplified using the tiling sequencing strategy which divided the whole mtGenome into 162 amplicons. All amplicons were successfully recovered. A phylogenetic analysis was performed to determine the accuracy of the PGM data, and which were compared to partial Sanger-based sequencing data. The average coverage of the PGM data were above 4000× in all case samples, and 99.86% concordance was observed using both sequencing methods. In conclusion, we demonstrate the ability to recover the complete mtGenome from bloodstains with relatively poor DNA quality by PGM. Moreover, the results are concordant with Sanger sequencing data. This new method has potential use in forensic practice.

Identification of aged bloodstains through mRNA profiling: Experiments results on selected markers of 30- and 50-year-old samples.

Remarkable progress has been made in recent years on the research of body fluid identification through messenger RNA(mRNA) profiling. In order to examine the viability of mRNA profiling as a method to identify aged bloodstains, this study tested two groups of bloodstain samples, dated 30 years and 50 years back respectively, on seven blood specific markers, i.e. HBB, HBA, GYPA, CD93, ALAS2, SPTB (91bp and 247bp primers), and PBGD. Test results indicate that HBA and HBB are the most stable markers in aged bloodstains, returning positive results in over 80% of the 50-year-old samples and over 90% of the 30-year-old samples. This finding proves mRNA profiling an effective way of identifying aged bloodstains.

Y-chromosomal haplotyping of single sperm cells isolated from semen mixtures - a successful identification of three perpetrators in a multi-suspect sexual assault case.

AIM: To obtain individual Y-short tandem repeat (STR) profiles in a multi-suspect sexual assault case. Methods. We used laser cut microdissection to capture the single sperm cell in the multi-contributor semen sample, combined with the low volume polymerase chain reaction (LV-PCR) method to genotype the single sperm cell profiles using the Yfiler(®) kit. Consensus DNA profiles were generated from 5 replicate experiments. Results. Ninety-four parallel LV-PCRs were performed and 41 reactions (44%) produced Y-STR profiles with more than nine loci. Three individual Y-STR profiles were successfully obtained. Conclusion. The three Y haplotype units matched three known perpetrators' genotypes. Our results showed that single sperm cells Y-STR analysis was a powerful method for analyzing multi-donor semen mixture sample.

New cell separation technique for the isolation and analysis of cells from biological mixtures in forensic caseworks.

AIM: To isolate mucosal cells of the perpetrator in a sexual assault case from a complex mixture of his mucosal cells and the victim's skin by micromanipulation prior to genomic analysis. METHODS: To capture and analyze mucosal cells we used the micromanipulation with on-chip low volume polymerase chain reaction (LV-PCR). Consensus DNA profiles were generated from 5 replicate experiments. RESULTS AND CONCLUSIONS: We validated the use of micromanipulation with on-chip LV-PCR for genomic analysis of complex biological mixtures in a fatal rape case. The perpetrator's mucosal cells were captured from nipple swabs of the victim, and a single-source DNA profile was generated from cell mixtures. These data suggest that micromanipulation with on-chip LV-PCR is an effective forensic tool for the analysis of specific cells from complex samples.

10 Y-STRs haplotypes in Chinese.

Haplotypes of DYS389I, YS389II, DYS439, DYS438, DYS392, DYS393, DYS19, DYS390, DYS391, DYS385 were determined from 136 unrelated Chinese male individuals.