Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification.

OBJECTIVES: To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification. METHODS: The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained. RESULTS: There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/µL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/µL, and the discrimination accuracy was 100%. CONCLUSIONS: In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.

Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid.

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C10-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s-1 mM-1 to 119 s-1 mM-1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L-1 day-1 and 160 g L-1 day-1, respectively.

Optimization and validation of analysis method based on 27-plex SNP panel for ancestry inference.

Anthropology generally divides the individuals into the East Asian Mongolia race, European Caucasian race and African Nigro race. The 27-plex single nucleotide polymorphism (SNP) panel for ancestry information has been established to differentiate samples from East Asian, European, African and admixture populations of East Asian and European origin by genotyping and ancestry inference. To infer ancestry for unknown individuals, we established an optimized analysis pipeline based on the likelihood ratio, ancestry component and individual ancestry assignment. Four samples from East Asian, European, African and admixture populations of East Asian and European origin were tested using the optimized analysis pipeline. Cross validation within basic referential database and validation of 1 010 test samples were both used to evaluate the inference process. The results showed that accuracy of the method was higher than 99% in East Asia, Europe, Africa and admixture populations. The inference method can characterize the ancestry information of DNA donors, and has important practical application values in the field of human molecular and forensic genetics.

Comparison of preprocessing methods and storage times for touch DNA samples.

AIM: To select appropriate preprocessing methods for different substrates by comparing the effects of four different preprocessing methods on touch DNA samples and to determine the effect of various storage times on the results of touch DNA sample analysis. METHOD: Hand touch DNA samples were used to investigate the detection and inspection results of DNA on different substrates. Four preprocessing methods, including the direct cutting method, stubbing procedure, double swab technique, and vacuum cleaner method, were used in this study. DNA was extracted from mock samples with four different preprocessing methods. The best preprocess protocol determined from the study was further used to compare performance after various storage times. DNA extracted from all samples was quantified and amplified using standard procedures. RESULTS: The amounts of DNA and the number of alleles detected on the porous substrates were greater than those on the non-porous substrates. The performances of the four preprocessing methods varied with different substrates. The direct cutting method displayed advantages for porous substrates, and the vacuum cleaner method was advantageous for non-porous substrates. No significant degradation trend was observed as the storage times increased. CONCLUSION: Different substrates require the use of different preprocessing method in order to obtain the highest DNA amount and allele number from touch DNA samples. This study provides a theoretical basis for explorations of touch DNA samples and may be used as a reference when dealing with touch DNA samples in case work.

A panel of 74 AISNPs: Improved ancestry inference within Eastern Asia.

Many ancestry informative SNP (AISNP) panels have been published. Ancestry resolution in them varies from three to eight continental clusters of populations depending on the panel used. However, none of these panels differentiates well among East Asian populations. To meet this need, we have developed a 74 AISNP panel after analyzing a much larger number of SNPs for Fst and allele frequency differences between two geographically close population groups within East Asia. The 74 AISNP panel can now distinguish at least 10 biogeographic groups of populations globally: Sub-Saharan Africa, North Africa, Europe, Southwest Asia, South Asia, North Asia, East Asia, Southeast Asia, Pacific and Americas. Compared with our previous 55-AISNP panel, Southeast Asia and North Asia are two newly assignable clusters. For individual ancestry assignment, the likelihood ratio and ancestry components were analyzed on a different set of 500 test individuals from 11 populations. All individuals from five of the test populations - Yoruba (YRI), European (CEU), Han Chinese in Henan (CHNH), Rondonian Surui (SUR) and Ticuna (TIC) - were assigned to their appropriate geographical regions unambiguously. For the other test populations, most of the individuals were assigned to their self-identified geographical regions with a certain degree of overlap with adjacent populations. These alternative ancestry components for each individual thus help give a clearer picture of the possible group origins of the individual. We have demonstrated that the new AISNP panel can achieve a deeper resolution of global ancestry.

Genetic structure and differentiation analysis of a Eurasian Uyghur population by use of 27 continental ancestry-informative SNPs.

Previously, we developed and validated a multiplex assay of 27 ancestry-informative markers (AIMs) for analyzing African (AFR), European (EUR), and East Asian (EAS) ancestry components. In this study, we typed and collectively analyzed a large Uyghur sample of 979 individuals to estimate the genetic coefficients of the 27 AIMs and investigate differentiation parameters between Uyghur and Han. The Uyghur allele frequencies ranged from 0.243 to 0.952, and heterozygosities ranged from 0.091 to 0.500. Values of F st 3 and I n 3 for EUR, Uyghur, and EAS ranged from 0.028 to 0.550 and 0.0002 to 0.345, respectively. The Uyghur population displays a substantial ancestry contribution of 50.3:49.7 (EUR:EAS) and was efficiently discriminated from Han Chinese with an accuracy of 99.285 %. All populations were clustered into AFR, EUR, EAS, and admixture groups of these three ancestries. Central Asian was obviously stratified from the other admixture populations of South Asians, North Asians, and the Americans. The 27 SNPs yield a circle with an average distance of 0.936 from the center (0, 0) in PCA analysis. Using this set, Chinese Uyghur and Han populations achieved accurate differentiation, and our updated genotype database (by citing 1000 Genomes data) of 43 worldwide populations is a useful resource for forensic applications and disease association studies.

A single-tube 27-plex SNP assay for estimating individual ancestry and admixture from three continents.

A single-tube multiplex assay of a small set of ancestry-informative markers (AIMs) for effectively estimating individual ancestry and admixture is an ideal forensic tool to trace the population origin of an unknown DNA sample. We present a newly developed 27-plex single nucleotide polymorphism (SNP) panel with highly robust and balanced differential power to perfectly assign individuals to African, European, and East Asian ancestries. Evaluating 968 previously described intercontinental AIMs from three HapMap population genotyping datasets (Yoruban in Ibadan, Nigeria (YRI); Utah residents with Northern and Western European ancestry from the Centre de'Etude du Polymorphism Humain (CEPH) collection (CEU); and Han Chinese in Beijing, China (CHB)), the best set of markers was selected on the basis of Hardy-Weinberg equilibrium (p > 0.00001), population-specific allele frequency (two of three δ values >0.5), according to linkage disequilibrium (r (2) < 0.2), and capable of being multiplexed in one tube and detected by capillary electrophoresis. The 27-SNP panel was first validated by assigning the ancestry of the 11 populations in the HapMap project. Then, we tested the 27-plex SNP assay with 1164 individuals from 17 additional populations. The results demonstrated that the SNP panel was successful for ancestry inference of individuals with African, European, and East Asian ancestry. Furthermore, the system performed well when inferring the admixture of Eurasians (EUR/EAS) after analyzing admixed populations from Xinjiang (Central Asian) as follows: Tajik (68:27), Uyghur (49:46), Kirgiz (40:57), and Kazak (36:60). For individual analyses, we interpreted each sample with a three-ancestry component percentage and a population match probability sequence. This multiplex assay is a convenient and cost-effective tool to assist in criminal investigations, as well as to correct for the effects of population stratification for case-control studies.

[Efficiency of 27-plex single nucleotide polymorphism multiplex system for ancestry inference in different populations].

OBJECTIVE: To validate the efficiency of 27-plex single nucleotide polymorphism (SNP) multiplex system for ancestry inference. METHODS: The 27-plex SNP system was validated for its sensitivity and species specificity. A total of 533 samples were collected from African, Southern Chinese Han, China's ethic minorities (Yi, Hui, Miao, Tibet, and Uygur), European, Central Asian, Western Asian, Southern Asian, Southeast Asian and South American populations for clustering analysis of the genotypes by citing 3 representative continental ancestral groups [East Asia (CHB), Europe (CEU), and Africa (YRI)] from HapMap database. RESULTS: The system sensitivity is 0.125 ng. Twenty and six genotypes were detected in chimpanzee and monkeys, respectively. Except in rs10496971, no more products were found in other animals. The system was capable of differentiating intercontinental populations but not of distinguishing between East Asian and Southeast Asian population or between Southern Chinese Han population and Chinese Ethnic populations (Hui, Miao, Yi and Tibet). This system achieved a 100% accuracy for intercontinental population source inference for 46 blind test samples. CONCLUSION: 27-plex SNPs multiplex system has a high sensitivity and species specificity and can correctly differentiate the ancestry origins of individuals from African, European and East Asian for criminal case investigation. But this system is not capable of distinguishing subpopulation groups and more specific ancestry-informative markers are needed to improve its recognition of Southeast Asian and Chinese ethnic populations.