High-throughput single nucleotide polymorphism(SNP) arrays have emerged as essential genotyping tools,significantly accelerating breeding programs and advancing basic research.In this study,a high-throughput 10K SNP g...High-throughput single nucleotide polymorphism(SNP) arrays have emerged as essential genotyping tools,significantly accelerating breeding programs and advancing basic research.In this study,a high-throughput 10K SNP genotyping array for wax gourd was developed using genotyping by target sequencing(GBTS),featuring 10,722 SNPs evenly distributed across all 12 chromosomes,including 278 functional loci associated with key economic traits.To demonstrate its utility,genetic distances among 19 elite inbred lines were calculated from SNP data and correlated with heterosis for single fruit weight.The results revealed that greater genetic distance was associated with higher middle parent heterosis(MPH) for single fruit weight.Furthermore,56 commercial wax gourd cultivars collected from eight regions were selected and genotyped.Population structure analysis,phylogenetic analysis,and principal component analysis(PCA) collectively indicated that these cultivars fall into two major groups.Group I,comprising black or dark green skinned wax gourds,exhibited lower genetic diversity than Group II,which includes green or light green skinned varieties,reflecting shorter genetic distances within Group I.Finally,60 polymorphic SNPs were used to construct DNA fingerprints for distinguishing the 56 cultivars.As the first high-throughput genotyping platform for wax gourd,this SNP array provides an effective and powerful tool for genetic analysis.展开更多
Genotyping by target sequencing(GBTS)integrates the advantages of silicon-based technology(high stability and reliability)and genotyping by sequencing(high flexibility and cost-effectiveness).However,GBTS panels are n...Genotyping by target sequencing(GBTS)integrates the advantages of silicon-based technology(high stability and reliability)and genotyping by sequencing(high flexibility and cost-effectiveness).However,GBTS panels are not currently available in pigs.In this study,based on GBTS technology,we first developed a 50K panel,including 52,000 single-nucleotide polymorphisms(SNPs),in pigs,designated GBTS50K.A total of 6,032 individuals of Large White,Landrace,and Duroc pigs from 10 breeding farms were used to assess the newly developed GBTS50K.Our results showed that GBTS50K obtained a high genotyping ability,the SNP and individual call rates of GBTS50K were 0.997–0.998,and the average consistency rate and genotyping correlation coefficient were 0.997 and 0.993,respectively,in replicate samples.We also evaluated the efficiencies of GBTS50K in the application of population genetic structure analysis,selection signature detection,genome-wide association studies(GWAS),genotyped imputation,genetic selection(GS),etc.The results indicate that GBTS50K is plausible and powerful in genetic analysis and molecular breeding.For example,GBTS50K could gain higher accuracies than the current popular GGP-Porcine bead chip in genomic selection on 2 important traits of backfat thickness at 100 kg and days to 100 kg in pigs.Particularly,due to the multiple SNPs(mSNPs),GBTS50K generated 100K qualified SNPs without increasing genotyping cost,and our results showed that the haplotype-based method can further improve the accuracies of genomic selection on growth and reproduction traits by 2 to 6%.Our study showed that GBTS50K could be a powerful tool for underlying genetic architecture and molecular breeding in pigs,and it is also helpful for developing SNP panels for other farm animals.展开更多
Accurate genotyping and prognosis of glioma patients present significant clinical challenges,often dependent on subjective judgement and insufficient scientific evidence.This study aims to develop a robust,noninvasive...Accurate genotyping and prognosis of glioma patients present significant clinical challenges,often dependent on subjective judgement and insufficient scientific evidence.This study aims to develop a robust,noninvasive preoperative multi-modal MRIbased transformer learning model to predict IDH genotyping and glioma prognosis.This multi-centre study included 563 glioma patients to develop an interpretable classification model utilising various preoperative imaging sequences,including T1-weighted,T2-weighted,fluid-attenuated inversion recovery,contrast-enhanced T1-weighted,and diffusion-weighted imaging.The model employs a multi-task learning framework to extract and fuse radiomic,deep learning,and clinical features for IDH genotyping and glioma prognosis.Additionally,a multi-modal transformer strategy is integrated to analyse structural and functional MRI,thereby enhancing model performance.Experimental results indicate that the model demonstrates superior performance,surpassing previous research and other state-of-the-art methods.The model achieves an AUC of 91.40% in the IDH genotyping task and 93.37% in the glioma prognosis task.Group analysis reveals that the model exhibits higher sensitivity to IDH-mutant cases and more accurately identifies low-risk groups compared to medium-or high-risk groups.This study aims to achieve accurate IDH genotyping and glioma prognosis through effective classification method,offering valuable diagnostic insights for clinical practice and expediting treatment decisions.展开更多
Fungi play crucial roles in nutrient acquisition,plant growth promotion,and the enhancement of resistance to both abiotic and biotic stresses.However,studies on the fungal communities associated with peas (Pisum sativ...Fungi play crucial roles in nutrient acquisition,plant growth promotion,and the enhancement of resistance to both abiotic and biotic stresses.However,studies on the fungal communities associated with peas (Pisum sativum L.) remain limited.In this study,we systematically investigated the ecological effects of host niches (soil,root,stem,leaf,and pod) and genotypes on the diversity and composition of fungal communities in peas using a multi-level approach that encompassed pattern recognition (β-diversity decomposition),mechanism validation (neutral community model testing),and dynamic tracking methods (migration pathway source-tracking).The results revealed that the dominant fungal phyla across niches and genotypes were Ascomycota,Basidiomycota,and Mortierellomycota,and the community structures of the soil–plant continuum were primarily determined by the pea niches rather than genotypes.β-diversity decomposition was largely attributed to species replacement rather than richness differences,indicating strong niche specificity and microbial replacement across microhabitats.Neutral model analysis revealed that stochastic processes influenced genotypeassociated communities,while deterministic processes played a dominant role in niche-based community assembly.Source-tracking analysis identified niche-to-niche fungal migration,with Erysiphe,Fusarium,Cephaliophora,Ascobolus,Alternaria,and Aspergillus as the key genera.Migration rates from exogenous to endogenous niches were low (1.3–61.5%),whereas those within exogenous (64.4–83.7%) or endogenous (73.9–96.4%) compartments were much higher,suggesting that the pea epidermis acts as a selective barrier that filters and enriches microbial communities prior to internal colonization.This study provides comprehensive insights into the mechanisms of host filtering,enrichment and microbial sourcing,which increases our understanding of the assembly rules of the pea-associated fungal microbiome.展开更多
Urinary tract infections(UTIs)are among the most prevalent pediatric bacterial infections,and undertreated episodes may lead to renal scarring,hypertension,or chronic kidney disease.Multidrug-resistant(MDR)Enterobacte...Urinary tract infections(UTIs)are among the most prevalent pediatric bacterial infections,and undertreated episodes may lead to renal scarring,hypertension,or chronic kidney disease.Multidrug-resistant(MDR)Enterobacterales have been increasingly reported in children,with higher rates in Asian and Middle Eastern settings than in high-income countries[1,2].展开更多
Two microsateUite DNA loci were used to trace the pedigree structure of six families in the shrimp Fenneropenaeus chinensis. Four of the families were natural mating, and the others were mated by artificial inseminati...Two microsateUite DNA loci were used to trace the pedigree structure of six families in the shrimp Fenneropenaeus chinensis. Four of the families were natural mating, and the others were mated by artificial insemination. Eleven alleles were acquired at two microsatellite DNA loci （locus RS0622 and locus EN0033 ） by investigating 145 offsprings and 11 parents. Five alleles were acquired from locus RS0622 and six from locus EN0033. As analyzed, the gene frequencies were between 0. 024 1 and 0. 493 1, the heterozygosity was 0.652 2 and 0.688 8, and the polymorphism information content （PIC） was 0.585 7 and 0.652 9 for the locus RS0622 and the locus EN0033, respectively. Twenty-three genotypes were detected and the genotypes of the losing parents were also inferred. The pedigrees of three F1 and three F2 generations were determined by matching the genotype at each locus.展开更多
AIM:To evaluate the impact of mass vaccination against the hepatitis B virus (HBV) in Egypt,and to search for vaccinee asymptomatic breakthrough HBV infection and its genotype.METHODS:Seven hundred serum samples from ...AIM:To evaluate the impact of mass vaccination against the hepatitis B virus (HBV) in Egypt,and to search for vaccinee asymptomatic breakthrough HBV infection and its genotype.METHODS:Seven hundred serum samples from vaccinated children and adults (aged 2-47 years) were used for quantitative and qualitative detection of HBsAb by ELISA.Three hundred and sixty serum samples representing undetectable or low or high HBsAb were screened for markers of active HBV infection (HBsAg,HBcAb (IgG) and HBeAb by ELISA,plus HBsAg by AxSYM) and HBV-DNA genotyping by nested multiplex PCR and by DNA sequencing.RESULTS:It was found that 65% of children aged 2-4 years,and 20.5% aged 4-13 years,as well as 45% adults were good responders to HBV vaccination mounting protective level HBsAb.Poor responders were 28%,59.5% and 34%,and non-responders were 7%,20% and 21% respectively,in the three studied groups.Markers of asymptomatic HBV infections were HBsAg detected by ELISA in 2.5% vs 11.39% by AxSYM.Other markers were HBcAb (IgG) in 1.38%,HBeAb in 0.83%,and HBV-DNA in 7.8%.All had HBV genotype E infection.CONCLUSION:It is concluded that HBV vaccine is efficient in controlling HBV infection among children and adults.The vaccine breakthrough infection was by HBV genotype E.A booster dose of vaccine is recommended,probably four years after initial vaccination.展开更多
An improved allele-specific PCR（AS-PCR） approach was applied to investigating -55C/T polymorphism in promoter region of the uncoupling protein 3（UCP3）gene. AS-PCR is a competitive PCR method which is based on posi...An improved allele-specific PCR（AS-PCR） approach was applied to investigating -55C/T polymorphism in promoter region of the uncoupling protein 3（UCP3）gene. AS-PCR is a competitive PCR method which is based on positioning the 3＇ base of a PCR primer to match one single nucleotide polymorphism（SNP） allele and accurately extend only the correctly matched primer. But it is limited in use because of its poor specificity. In this study, we improved the specificity of AS-PCR by introducing additional mismatch at the penultimate base of 3＇ end of AS-PCR primer in combination with decreasing the level of dNTP in the reaction mixture. Sensitivity, specificity and reliability of this method were assessed for both simple plasmid model and complex human genomic SNP targets. The -55C/T（rs1800849） polymorphisrn of the UCP3 gene was analyzed via this AS-PCR and restriction fragment length polymorphism（RFLP）, the latter was used as a gold standard. The results suggest that the increase in AS-PCR discrimination with this method should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping application.展开更多
Oculocutaneous albinism（OCA） is an autosomal recessive disorder characterized by hypopigmentation in eyes,hair and skin,accompanied with vision loss.Currently,six genes have been identified as causative genes for no...Oculocutaneous albinism（OCA） is an autosomal recessive disorder characterized by hypopigmentation in eyes,hair and skin,accompanied with vision loss.Currently,six genes have been identified as causative genes for non-syndromic OCA（OCA-1w4,6,7）,and ten genes for syndromic OCA（HPS-1e9,CHS-1）.Genetic counseling of 51 Chinese OCA families（39 OCA-1 with mutations in the TYR gene,6 OCA-2 with mutations in the OCA2 gene,4 OCA-4 with mutations in the SLC45A2 gene,1 HPS-1（Hermanskye Pudlak syndrome-1） with mutation in the HPS1 gene,and 1 mixed OCA-1 and OCA-4） led us to perform the prenatal genetic testing of OCA using amniotic fluid cells through the implementation of our optimized strategy.In our cohort,eleven previously unidentified alleles（PUAs）（5 in TYR,2 in OCA2,and 4 in SLC45A2） were found.Three missense PUAs（p.C112 R,p.H363 R and p.G379 V of TYR） and one in-frame deletional PUA（p.S222 del of SLC24A5） led to fetuses with OCA when co-inherited with other disease causative alleles.Three PUAs（p.P152 H and p.W272 X of TYR,p.A486 T of SLC24A5） identified in the OCA probands did not co-transmit with known pathological alleles and thus gave rise to unaffected fetuses.Four PUAs（p.Q83 X and p.A658 T of TYR,p.G161 R and p.G366 R of SLC24A5） did not transmit to the unaffected fetuses.In addition,the in vitro transfection assays showed that the p.S192 Y variant of TYR produced less pigment compared to the wild-type allele.A fetus with a digenic carrier of OCA-1 and OCA-4 was unaffected.In combination with functional assays,the family inheritance pattern is useful for the evaluation of pathogenicity of PUAs and genetic counseling of OCA.展开更多
We have developed a novel dual enzyme chemistry called rhAmp®SNP genotyping based on RNase H2-dependent PCR (rhPCR) that provides high signal and specificity for SNP analysis. rhAmp SNP genotyping combines a u...We have developed a novel dual enzyme chemistry called rhAmp®SNP genotyping based on RNase H2-dependent PCR (rhPCR) that provides high signal and specificity for SNP analysis. rhAmp SNP genotyping combines a unique two-enzyme system with 3’ end blocked DNA-RNA hybrid primers to interrogate SNP loci. Activation of the blocked primers occurs upon hybridization to its perfectly matched target, which eliminates or greatly reduces primer dimers. A thermostable hot-start RNase H2 cleaves the primer immediately 5’ of the ribose sugar, releasing the blocking group and allowing primer extension. PCR specificity is further improved with the use of a mutant Taq DNA polymerase, resulting in improved allelic discrimination. Signal generation is obtained using a universal reporter system which requires only two reporter probes for any bi-allelic SNP. 1000 randomly selected SNPs were chosen to validate the 95% design rate of the design pipeline. A subsampling of 130 human SNP targets was tested and achieved a 98% call rate, and 99% call accuracy. rhAmp SNP genotyping assays are compatible with various qPCR instruments including QuantStudioTM 7 Flex, CFX384TM, IntelliQube®, and Biomark HDTM. In comparison to TaqMan®, rhAmp SNP genotyping assays show higher signal (Rn) and greater cluster separation, resulting in more reliable SNP genotyping performance. The rhAmp SNP genotyping solution is suited for high-throughput SNP genotyping applications in humans and plants.展开更多
Background:Genotyping by sequencing(GBS)is a robust method to genotype markers.Many factors can influence the genotyping quality.One is that heterozygous genotypes could be wrongly genotyped as homozygotes,dependent o...Background:Genotyping by sequencing(GBS)is a robust method to genotype markers.Many factors can influence the genotyping quality.One is that heterozygous genotypes could be wrongly genotyped as homozygotes,dependent on the genotyping depths.In this study,a method correcting this type of genotyping error was demonstrated.The efficiency of this correction method and its effect on genomic prediction were assessed using simulated data of livestock populations.Results:Chip array(Chip)and four depths of GBS data was simulated.After quality control(call rate≥0.8 and MAF≥0.01),the remaining number of Chip and GBS SNPs were both approximately 7,000,averaged over 10 replicates.GBS genotypes were corrected with the proposed method.The reliability of genomic prediction was calculated using GBS,corrected GBS(GBSc),true genotypes for the GBS loci(GBSr)and Chip data.The results showed that GBSc had higher rates of correct genotype calls and higher correlations with true genotypes than GBS.For genomic prediction,using Chip data resulted in the highest reliability.As the depth increased to 10,the prediction reliabilities using GBS and GBSc data approached those using true GBS data.The reliabilities of genomic prediction using GBSc data were 0.604,0.672,0.684 and 0.704 after genomic correction,with the improved values of 0.013,0.009,0.006 and 0.001 at depth=2,4,5 and 10,respectively.Conclusions:The current study showed that a correction method for GBS data increased the genotype accuracies and,consequently,improved genomic predictions.These results suggest that a correction of GBS genotype is necessary,especially for the GBS data with low depths.展开更多
Common variants explain little of the variance of most common disease, prompting large-scale sequencing studies to understand the contribution of rare variants to these diseases. Imputation of rare variants from genom...Common variants explain little of the variance of most common disease, prompting large-scale sequencing studies to understand the contribution of rare variants to these diseases. Imputation of rare variants from genome-wide genotypic arrays offers a cost-efficient strategy to achieve necessary sample sizes required for adequate statistical power. To estimate the performance of imputation of rare variants, we imputed 153 individuals, each of whom was genotyped on 3 different genotype arrays including 317k, 610k and 1 million single nucleotide polymorphisms （SNPs）, to two different reference panels： HapMap2 and 1000 Genomes pilot March 2010 release （1KGpilot） by using IMPUTE version 2. We found that more than 94% and 84% of all SNPs yield acceptable accuracy （info 〉 0.4） in HapMap2 and 1KGpilot-based imputation, respectively. For rare variants （minor allele frequency （MAF） 〈5%）, the proportion of well- imputed SNPs increased as the MAF increased from 0.3% to 5% across all 3 genome-wide association study （GWAS） datasets. The proportion of well-imputed SNPs was 69%, 60% and 49% for SNPs with a MAF from 0.3% to 5% for 1M, 610k and 317k, respectively. None of the very rare variants （MAF 〈 0.3%） were well imputed. We conclude that the imputation accuracy of rare variants increases with higher density of genome-wide genotyping arrays when the size of the reference panel is small. Variants with lower MAF are more difficult to impute. These findings have important implications in the design and replication of large-scale sequencing studies.展开更多
The Genome Analysis Toolkit(GATK) is a popular set of programs for discovering and genotyping variants from next-generation sequencing data.The current GATK recommendation for RNA sequencing(RNA-seq) is to perform var...The Genome Analysis Toolkit(GATK) is a popular set of programs for discovering and genotyping variants from next-generation sequencing data.The current GATK recommendation for RNA sequencing(RNA-seq) is to perform variant calling from individual samples,with the drawback that only variable positions are reported.Versions 3.0 and above of GATK offer the possibility of calling DNA variants on cohorts of samples using the HaplotypeCaller algorithm in Genomic Variant Call Format(GVCF) mode.Using this approach,variants are called individually on each sample,generating one GVCF file per sample that lists genotype likelihoods and their genome annotations.In a second step,variants are called from the GVCF files through a joint genotyping analysis.This strategy is more flexible and reduces computational challenges in comparison to the traditional joint discovery workflow.Using a GVCF workflow for mining SNP in RNA-seq data provides substantial advantages,including reporting homozygous genotypes for the reference allele as well as missing data.Taking advantage of RNA-seq data derived from primary macrophages isolated from 50 cows,the GATK joint genotyping method for calling variants on RNA-seq data was validated by comparing this approach to a so-called "per-sample" method.In addition,pair-wise comparisons of the two methods were performed to evaluate their respective sensitivity,precision and accuracy using DNA genotypes from a companion study including the same 50 cows genotyped using either genotyping-by-sequencing or with the Bovine SNP50 Beadchip(imputed to the Bovine high density).Results indicate that both approaches are very close in their capacity of detecting reference variants and that the joint genotyping method is more sensitive than the per-sample method.Given that the joint genotyping method is more flexible and technically easier,we recommend this approach for variant calling in RNA-seq experiments.展开更多
Objective To evaluate four candidate variable number tandem repeat （VNTR） loci for genotyping Mycobacterium tuberculosis complex strains. Methods Genomic sequences for two M. tuberculosis strains （CCDC5079 and CCDC...Objective To evaluate four candidate variable number tandem repeat （VNTR） loci for genotyping Mycobacterium tuberculosis complex strains. Methods Genomic sequences for two M. tuberculosis strains （CCDC5079 and CCDC5180） were generated, and using published sequence data, four candidate VNTR loci were identified. The VNTRs were used to genotype 225 Chinese clinical M. tuberculosis complex strains. The discriminatory power of the VNTRs was evaluated using BioNumerics 5.0 software. Results The Hunter-Gaston Index （HGI） for BJ1, BJ2, BJ3, and BJ4 loci was 0.634, 0.917, 0.697, and 0.910, respectively. Combining all four loci gave an HGI value of 0.995, thus confirming that the genotyping had good discriminatory power. The HGI values for BJ1, BJ2, BJ3, and BJ4, obtained from Beijing family strain genotyping, were 0.447, 0.878, 0.315, and 0.850, respectively. Combining all four loci produced an HGI value of 0.988 for genotyping the Beijing family strains. We observed unique patterns for M. boris and M. africanum strains from the four loci. Conclusion We have shown that the four VNTR loci can be successfully used for genotyping M. tuberculosis complex strains. Notably, these new loci may provide additional information about Chinese M. tuberculosis isolates than that currently afforded by established VNTR loci typing.展开更多
Objective Human Lyme Borreliosis （LB）, which is caused by Borrefia burgdorferi sensu lato （B. burgdorferi）, has been identified as a major arthropod-borne infectious disease in China. We aimed to develop a multipl...Objective Human Lyme Borreliosis （LB）, which is caused by Borrefia burgdorferi sensu lato （B. burgdorferi）, has been identified as a major arthropod-borne infectious disease in China. We aimed to develop a multiple locus variable-number tandem repeat （VNTR） analysis （MLVA） assay for the genotyping of Borrelia burgdorJ：eri strains detected in China. Methods B. garinii PBi complete 904.246 kb chromosome and two plasmids （cp26 and Ip54） were screened by using Tandem Repeats Finder program for getting potential VNTR loci, the potential VNTR loci were analyzed and identified with PCR and the VNTR loci data were analyzed and MLVA clustering tree were constrcted by using the categorical coefficient and the unweighted pair-group method with arithmetic means （UPGMA）. Results We identified 5 new VNTR loci through analyzing 47 potential VNTR loci. We used the MLVA protocol to analyse 101 B. burgdorferi strains detected in China and finally identified 51 unique genotypes in 4 major clusters including B. burgdorferi sensu stricto （B.b.s.s）, B. garinii, B. a[zelii, and B. valaisiana, consistent with the current MLSA phylogeny studies. The allele numbers of VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5 were 7, 3, 9, 7, and 6. The Hunter-Gaston index （HGI） of five VNTR loci were 0.79, 0.22, 0.77, 0.71, and 0.67, respectively. The combined HGI of five VNTR loci was 0.96. Clustering of the strains of Xinjiang, Inner Mongolia and Heilongjiang was confirmed, and this situation was consistent with the close geographical distribution of those provinces. Conclusion The MLVA protocol esytablished in this study is easy and can show strains＇ phylogenetic relationships to distinguish the strains of Borrelia species. It is useful for further phylogenetic and epidemiological analyses of Borrelia strains.展开更多
The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover...The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout（KO） or knockin（KI） animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA（gDNA） followed by an automated capillary electrophoresis step which reveals a heteroduplexes（HD） signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.展开更多
In modern wildlife ecological research,feces is the most common non-invasive source of DNA obtained in the field and polymerase chain reaction(PCR) technology based on microsatellite markers is used to mine genetic in...In modern wildlife ecological research,feces is the most common non-invasive source of DNA obtained in the field and polymerase chain reaction(PCR) technology based on microsatellite markers is used to mine genetic information contained within.This is especially the case for endangered species.However,there are risks associated with this genotyping method because of the poor quality of fecal DNA.In this study,we assessed genotyping risk across 12 microsatellite loci commonly used in previous tiger studies using blood and fecal DNA from captive Amur tigers(Panthera tigris altaica).To begin,we developed an index termed the accumulated matching rate of genotypes(R)between positive DNA(blood samples) and fecal DNA to explore the correct genotyping probability of a certain microsatellite locus.We found that different microsatelliteloci had different genotyping risks and required different PCR amplification protocols.The genotyping errors we detected altered population genetic parameters and potentially impact subsequent analyses.Based on these findings,we recommend that:(1) four loci(E7,Fca094,Pti007 and Pti010) of 12 loci are not suitable for Amur tiger genetic research because of a low Rand difficulty reaching a stable status;(2) the Rof the 12 microsatellite loci plateaued differently,and considering limited budgets,amplification times of some loci could be increased when using fecal samples; and(3) future genetic analysis of wild Amur tigers should be corrected by genotyping error rates(1-R).展开更多
Rapeseed(Brassica napus)is an oil crop grown worldwide,making it a key plant species in molecular breeding research.However,the complexity of its polyploid genome increases sequencing costs and reduces sequencing accu...Rapeseed(Brassica napus)is an oil crop grown worldwide,making it a key plant species in molecular breeding research.However,the complexity of its polyploid genome increases sequencing costs and reduces sequencing accuracy.Target capture coupled with high-throughput sequencing is an efficient approach for detecting genetic variation at genomic regions or loci of interest.In this study,588 resequenced accessions of rapeseed were used to develop a target capture sequencing SNP genotyping platform named BnaPan50T.The platform comprised 54,765,with 54,058 resequenced markers from the pan-genome,and 855 variant trait-associated markers for 12 agronomic traits.The capture quality of BnaPan50T was demonstrated well in 12 typical accessions.Compared with a conventional genotyping array,BnaPan50T has a high SNP density and a high proportion of SNPs in unique physical positions and in annotated functional genes,promising wide application.Target capture sequencing and wholegenome resequencing in 90 doubled-haploid lines yielded 60%specificity,78%uniformity within tenfold coverage range,and 93%genotyping accuracy for the platform.BnaPan50T was used to construct a genetic map for quantitative trait loci(QTL)mapping,identify 21 unique QTL,and predict several candidate genes for yield-related traits in multiple environments.A set of 132 core SNP loci was selected from BnaPan50T to construct DNA fingerprints and germplasm identification resources.This study provides genomics resources to support target capture sequencing,genetic analysis and genomic breeding of rapeseed.展开更多
To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical se...To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS： Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 （33%）, 41 （33%） and 45 （36%） samples, and genotype C in 76 （60%）, 76 （60%） and 81 （64%） samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real- time PCR was the rapidest and cheapest among the three assays. CONCLUSION： Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.展开更多
Background:Genotyping by sequencing(GBS)still has problems with missing genotypes.Imputation is important for using GBS for genomic predictions,especially for low depths,due to the large number of missing genotypes.Mi...Background:Genotyping by sequencing(GBS)still has problems with missing genotypes.Imputation is important for using GBS for genomic predictions,especially for low depths,due to the large number of missing genotypes.Minor allele frequency(MAF)is widely used as a marker data editing criteria for genomic predictions.In this study,three imputation methods(Beagle,IMPUTE2 and FImpute software)based on four MAF editing criteria were investigated with regard to imputation accuracy of missing genotypes and accuracy of genomic predictions,based on simulated data of livestock population.Results:Four MAFs(no MAF limit,MAF≥0.001,MAF≥0.01 and MAF≥0.03)were used for editing marker data before imputation.Beagle,IMPUTE2 and FImpute software were applied to impute the original GBS.Additionally,IMPUTE2 also imputed the expected genotype dosage after genotype correction(GcIM).The reliability of genomic predictions was calculated using GBS and imputed GBS data.The results showed that imputation accuracies were the same for the three imputation methods,except for the data of sequencing read depth(depth)=2,where FImpute had a slightly lower imputation accuracy than Beagle and IMPUTE2.GcIM was observed to be the best for all of the imputations at depth=4,5 and 10,but the worst for depth=2.For genomic prediction,retaining more SNPs with no MAF limit resulted in higher reliability.As the depth increased to 10,the prediction reliabilities approached those using true genotypes in the GBS loci.Beagle and IMPUTE2 had the largest increases in prediction reliability of 5 percentage points,and FImpute gained 3 percentage points at depth=2.The best prediction was observed at depth=4,5 and 10 using GcIM,but the worst prediction was also observed using GcIM at depth=2.Conclusions:The current study showed that imputation accuracies were relatively low for GBS with low depths and high for GBS with high depths.Imputation resulted in larger gains in the reliability of genomic predictions for GBS with lower depths.These results suggest that the application of IMPUTE2,based on a corrected GBS(GcIM)to improve genomic predictions for higher depths,and FImpute software could be a good alternative for routine imputation.展开更多
基金supported by the Science and Technology Talent Support Project of Hunan Province,China (2022TJ-N15)the Hunan Agricultural Science and Technology Innovation Fund,China (2024CX90 and 2024CX65)the Science and Technology Innovation Program of Hunan Province,China (2021NK1006)。
文摘High-throughput single nucleotide polymorphism(SNP) arrays have emerged as essential genotyping tools,significantly accelerating breeding programs and advancing basic research.In this study,a high-throughput 10K SNP genotyping array for wax gourd was developed using genotyping by target sequencing(GBTS),featuring 10,722 SNPs evenly distributed across all 12 chromosomes,including 278 functional loci associated with key economic traits.To demonstrate its utility,genetic distances among 19 elite inbred lines were calculated from SNP data and correlated with heterosis for single fruit weight.The results revealed that greater genetic distance was associated with higher middle parent heterosis(MPH) for single fruit weight.Furthermore,56 commercial wax gourd cultivars collected from eight regions were selected and genotyped.Population structure analysis,phylogenetic analysis,and principal component analysis(PCA) collectively indicated that these cultivars fall into two major groups.Group I,comprising black or dark green skinned wax gourds,exhibited lower genetic diversity than Group II,which includes green or light green skinned varieties,reflecting shorter genetic distances within Group I.Finally,60 polymorphic SNPs were used to construct DNA fingerprints for distinguishing the 56 cultivars.As the first high-throughput genotyping platform for wax gourd,this SNP array provides an effective and powerful tool for genetic analysis.
基金supported by the grants from the Key R&D Program of Shandong Province,China(2022LZGC003)the China Agriculture Research System of MOF and MARA(CARS-35)+1 种基金the National Key Research and Development Project of China(2019YFE0106800)the 2115 Talent Development Program of China Agricultural University。
文摘Genotyping by target sequencing(GBTS)integrates the advantages of silicon-based technology(high stability and reliability)and genotyping by sequencing(high flexibility and cost-effectiveness).However,GBTS panels are not currently available in pigs.In this study,based on GBTS technology,we first developed a 50K panel,including 52,000 single-nucleotide polymorphisms(SNPs),in pigs,designated GBTS50K.A total of 6,032 individuals of Large White,Landrace,and Duroc pigs from 10 breeding farms were used to assess the newly developed GBTS50K.Our results showed that GBTS50K obtained a high genotyping ability,the SNP and individual call rates of GBTS50K were 0.997–0.998,and the average consistency rate and genotyping correlation coefficient were 0.997 and 0.993,respectively,in replicate samples.We also evaluated the efficiencies of GBTS50K in the application of population genetic structure analysis,selection signature detection,genome-wide association studies(GWAS),genotyped imputation,genetic selection(GS),etc.The results indicate that GBTS50K is plausible and powerful in genetic analysis and molecular breeding.For example,GBTS50K could gain higher accuracies than the current popular GGP-Porcine bead chip in genomic selection on 2 important traits of backfat thickness at 100 kg and days to 100 kg in pigs.Particularly,due to the multiple SNPs(mSNPs),GBTS50K generated 100K qualified SNPs without increasing genotyping cost,and our results showed that the haplotype-based method can further improve the accuracies of genomic selection on growth and reproduction traits by 2 to 6%.Our study showed that GBTS50K could be a powerful tool for underlying genetic architecture and molecular breeding in pigs,and it is also helpful for developing SNP panels for other farm animals.
基金supported by the National Natural Science Foundation of China(Grants 82441022,82371934)Medical Science and Technology Research Project of Henan Province(SBGJ202101002)+1 种基金Joint Fund of Henan Province Science and Technology R&D Programme(225200810062)Henan Provincial Medical Science and Technology Research Joint Construction Project(LHGJ20240053,LHGJ20240036).
文摘Accurate genotyping and prognosis of glioma patients present significant clinical challenges,often dependent on subjective judgement and insufficient scientific evidence.This study aims to develop a robust,noninvasive preoperative multi-modal MRIbased transformer learning model to predict IDH genotyping and glioma prognosis.This multi-centre study included 563 glioma patients to develop an interpretable classification model utilising various preoperative imaging sequences,including T1-weighted,T2-weighted,fluid-attenuated inversion recovery,contrast-enhanced T1-weighted,and diffusion-weighted imaging.The model employs a multi-task learning framework to extract and fuse radiomic,deep learning,and clinical features for IDH genotyping and glioma prognosis.Additionally,a multi-modal transformer strategy is integrated to analyse structural and functional MRI,thereby enhancing model performance.Experimental results indicate that the model demonstrates superior performance,surpassing previous research and other state-of-the-art methods.The model achieves an AUC of 91.40% in the IDH genotyping task and 93.37% in the glioma prognosis task.Group analysis reveals that the model exhibits higher sensitivity to IDH-mutant cases and more accurately identifies low-risk groups compared to medium-or high-risk groups.This study aims to achieve accurate IDH genotyping and glioma prognosis through effective classification method,offering valuable diagnostic insights for clinical practice and expediting treatment decisions.
基金financial y supported by the National Key Research and Development Program of China (2023YFD1900902)the Joint Funds of the Zhejiang Provincial Natural Science Foundation of China (LLSSZ24C030001)+1 种基金the Earmarked Fund for China Agriculture Research System (CARS-08-G-09)sponsored by the K.C.Wong Magna Fund of Ningbo University,China。
文摘Fungi play crucial roles in nutrient acquisition,plant growth promotion,and the enhancement of resistance to both abiotic and biotic stresses.However,studies on the fungal communities associated with peas (Pisum sativum L.) remain limited.In this study,we systematically investigated the ecological effects of host niches (soil,root,stem,leaf,and pod) and genotypes on the diversity and composition of fungal communities in peas using a multi-level approach that encompassed pattern recognition (β-diversity decomposition),mechanism validation (neutral community model testing),and dynamic tracking methods (migration pathway source-tracking).The results revealed that the dominant fungal phyla across niches and genotypes were Ascomycota,Basidiomycota,and Mortierellomycota,and the community structures of the soil–plant continuum were primarily determined by the pea niches rather than genotypes.β-diversity decomposition was largely attributed to species replacement rather than richness differences,indicating strong niche specificity and microbial replacement across microhabitats.Neutral model analysis revealed that stochastic processes influenced genotypeassociated communities,while deterministic processes played a dominant role in niche-based community assembly.Source-tracking analysis identified niche-to-niche fungal migration,with Erysiphe,Fusarium,Cephaliophora,Ascobolus,Alternaria,and Aspergillus as the key genera.Migration rates from exogenous to endogenous niches were low (1.3–61.5%),whereas those within exogenous (64.4–83.7%) or endogenous (73.9–96.4%) compartments were much higher,suggesting that the pea epidermis acts as a selective barrier that filters and enriches microbial communities prior to internal colonization.This study provides comprehensive insights into the mechanisms of host filtering,enrichment and microbial sourcing,which increases our understanding of the assembly rules of the pea-associated fungal microbiome.
文摘Urinary tract infections(UTIs)are among the most prevalent pediatric bacterial infections,and undertreated episodes may lead to renal scarring,hypertension,or chronic kidney disease.Multidrug-resistant(MDR)Enterobacterales have been increasingly reported in children,with higher rates in Asian and Middle Eastern settings than in high-income countries[1,2].
基金the National High Technology Development Project of China under contract No. 2005AA603210 the National Natural Science Foundation of China under contract No. 30500378.
文摘Two microsateUite DNA loci were used to trace the pedigree structure of six families in the shrimp Fenneropenaeus chinensis. Four of the families were natural mating, and the others were mated by artificial insemination. Eleven alleles were acquired at two microsatellite DNA loci （locus RS0622 and locus EN0033 ） by investigating 145 offsprings and 11 parents. Five alleles were acquired from locus RS0622 and six from locus EN0033. As analyzed, the gene frequencies were between 0. 024 1 and 0. 493 1, the heterozygosity was 0.652 2 and 0.688 8, and the polymorphism information content （PIC） was 0.585 7 and 0.652 9 for the locus RS0622 and the locus EN0033, respectively. Twenty-three genotypes were detected and the genotypes of the losing parents were also inferred. The pedigrees of three F1 and three F2 generations were determined by matching the genotype at each locus.
基金Biotechnology and Genetic Engineering National Strategy- Academy for Science,Research and Biotechnology for their financial support to this project
文摘AIM:To evaluate the impact of mass vaccination against the hepatitis B virus (HBV) in Egypt,and to search for vaccinee asymptomatic breakthrough HBV infection and its genotype.METHODS:Seven hundred serum samples from vaccinated children and adults (aged 2-47 years) were used for quantitative and qualitative detection of HBsAb by ELISA.Three hundred and sixty serum samples representing undetectable or low or high HBsAb were screened for markers of active HBV infection (HBsAg,HBcAb (IgG) and HBeAb by ELISA,plus HBsAg by AxSYM) and HBV-DNA genotyping by nested multiplex PCR and by DNA sequencing.RESULTS:It was found that 65% of children aged 2-4 years,and 20.5% aged 4-13 years,as well as 45% adults were good responders to HBV vaccination mounting protective level HBsAb.Poor responders were 28%,59.5% and 34%,and non-responders were 7%,20% and 21% respectively,in the three studied groups.Markers of asymptomatic HBV infections were HBsAg detected by ELISA in 2.5% vs 11.39% by AxSYM.Other markers were HBcAb (IgG) in 1.38%,HBeAb in 0.83%,and HBV-DNA in 7.8%.All had HBV genotype E infection.CONCLUSION:It is concluded that HBV vaccine is efficient in controlling HBV infection among children and adults.The vaccine breakthrough infection was by HBV genotype E.A booster dose of vaccine is recommended,probably four years after initial vaccination.
基金Supported by the Major Project Fund of Jilin Provincial Science and Technology Department,China(No.20082123)Province-university Union Fund of Jilin Province,China(No.20082011)
文摘An improved allele-specific PCR（AS-PCR） approach was applied to investigating -55C/T polymorphism in promoter region of the uncoupling protein 3（UCP3）gene. AS-PCR is a competitive PCR method which is based on positioning the 3＇ base of a PCR primer to match one single nucleotide polymorphism（SNP） allele and accurately extend only the correctly matched primer. But it is limited in use because of its poor specificity. In this study, we improved the specificity of AS-PCR by introducing additional mismatch at the penultimate base of 3＇ end of AS-PCR primer in combination with decreasing the level of dNTP in the reaction mixture. Sensitivity, specificity and reliability of this method were assessed for both simple plasmid model and complex human genomic SNP targets. The -55C/T（rs1800849） polymorphisrn of the UCP3 gene was analyzed via this AS-PCR and restriction fragment length polymorphism（RFLP）, the latter was used as a gold standard. The results suggest that the increase in AS-PCR discrimination with this method should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping application.
基金partially supported by grants from the National Natural Science Foundation of China(Nos.81472871 and31230046)the Natural Science Foundation of Beijing(No.7132073)the State Key Laboratory of Molecular Developmental Biology of China(No.2013-MDB-KF-03)
文摘Oculocutaneous albinism（OCA） is an autosomal recessive disorder characterized by hypopigmentation in eyes,hair and skin,accompanied with vision loss.Currently,six genes have been identified as causative genes for non-syndromic OCA（OCA-1w4,6,7）,and ten genes for syndromic OCA（HPS-1e9,CHS-1）.Genetic counseling of 51 Chinese OCA families（39 OCA-1 with mutations in the TYR gene,6 OCA-2 with mutations in the OCA2 gene,4 OCA-4 with mutations in the SLC45A2 gene,1 HPS-1（Hermanskye Pudlak syndrome-1） with mutation in the HPS1 gene,and 1 mixed OCA-1 and OCA-4） led us to perform the prenatal genetic testing of OCA using amniotic fluid cells through the implementation of our optimized strategy.In our cohort,eleven previously unidentified alleles（PUAs）（5 in TYR,2 in OCA2,and 4 in SLC45A2） were found.Three missense PUAs（p.C112 R,p.H363 R and p.G379 V of TYR） and one in-frame deletional PUA（p.S222 del of SLC24A5） led to fetuses with OCA when co-inherited with other disease causative alleles.Three PUAs（p.P152 H and p.W272 X of TYR,p.A486 T of SLC24A5） identified in the OCA probands did not co-transmit with known pathological alleles and thus gave rise to unaffected fetuses.Four PUAs（p.Q83 X and p.A658 T of TYR,p.G161 R and p.G366 R of SLC24A5） did not transmit to the unaffected fetuses.In addition,the in vitro transfection assays showed that the p.S192 Y variant of TYR produced less pigment compared to the wild-type allele.A fetus with a digenic carrier of OCA-1 and OCA-4 was unaffected.In combination with functional assays,the family inheritance pattern is useful for the evaluation of pathogenicity of PUAs and genetic counseling of OCA.
文摘We have developed a novel dual enzyme chemistry called rhAmp®SNP genotyping based on RNase H2-dependent PCR (rhPCR) that provides high signal and specificity for SNP analysis. rhAmp SNP genotyping combines a unique two-enzyme system with 3’ end blocked DNA-RNA hybrid primers to interrogate SNP loci. Activation of the blocked primers occurs upon hybridization to its perfectly matched target, which eliminates or greatly reduces primer dimers. A thermostable hot-start RNase H2 cleaves the primer immediately 5’ of the ribose sugar, releasing the blocking group and allowing primer extension. PCR specificity is further improved with the use of a mutant Taq DNA polymerase, resulting in improved allelic discrimination. Signal generation is obtained using a universal reporter system which requires only two reporter probes for any bi-allelic SNP. 1000 randomly selected SNPs were chosen to validate the 95% design rate of the design pipeline. A subsampling of 130 human SNP targets was tested and achieved a 98% call rate, and 99% call accuracy. rhAmp SNP genotyping assays are compatible with various qPCR instruments including QuantStudioTM 7 Flex, CFX384TM, IntelliQube®, and Biomark HDTM. In comparison to TaqMan®, rhAmp SNP genotyping assays show higher signal (Rn) and greater cluster separation, resulting in more reliable SNP genotyping performance. The rhAmp SNP genotyping solution is suited for high-throughput SNP genotyping applications in humans and plants.
基金supported by the Genomic Selection in PlantsAnimals(GenSAP)research project financed by the Danish Council of Strategic Research(Aarhus,Denmark)the scholarship provided by the China Scholarship Council(CSC)
文摘Background:Genotyping by sequencing(GBS)is a robust method to genotype markers.Many factors can influence the genotyping quality.One is that heterozygous genotypes could be wrongly genotyped as homozygotes,dependent on the genotyping depths.In this study,a method correcting this type of genotyping error was demonstrated.The efficiency of this correction method and its effect on genomic prediction were assessed using simulated data of livestock populations.Results:Chip array(Chip)and four depths of GBS data was simulated.After quality control(call rate≥0.8 and MAF≥0.01),the remaining number of Chip and GBS SNPs were both approximately 7,000,averaged over 10 replicates.GBS genotypes were corrected with the proposed method.The reliability of genomic prediction was calculated using GBS,corrected GBS(GBSc),true genotypes for the GBS loci(GBSr)and Chip data.The results showed that GBSc had higher rates of correct genotype calls and higher correlations with true genotypes than GBS.For genomic prediction,using Chip data resulted in the highest reliability.As the depth increased to 10,the prediction reliabilities using GBS and GBSc data approached those using true GBS data.The reliabilities of genomic prediction using GBSc data were 0.604,0.672,0.684 and 0.704 after genomic correction,with the improved values of 0.013,0.009,0.006 and 0.001 at depth=2,4,5 and 10,respectively.Conclusions:The current study showed that a correction method for GBS data increased the genotype accuracies and,consequently,improved genomic predictions.These results suggest that a correction of GBS genotype is necessary,especially for the GBS data with low depths.
文摘Common variants explain little of the variance of most common disease, prompting large-scale sequencing studies to understand the contribution of rare variants to these diseases. Imputation of rare variants from genome-wide genotypic arrays offers a cost-efficient strategy to achieve necessary sample sizes required for adequate statistical power. To estimate the performance of imputation of rare variants, we imputed 153 individuals, each of whom was genotyped on 3 different genotype arrays including 317k, 610k and 1 million single nucleotide polymorphisms （SNPs）, to two different reference panels： HapMap2 and 1000 Genomes pilot March 2010 release （1KGpilot） by using IMPUTE version 2. We found that more than 94% and 84% of all SNPs yield acceptable accuracy （info 〉 0.4） in HapMap2 and 1KGpilot-based imputation, respectively. For rare variants （minor allele frequency （MAF） 〈5%）, the proportion of well- imputed SNPs increased as the MAF increased from 0.3% to 5% across all 3 genome-wide association study （GWAS） datasets. The proportion of well-imputed SNPs was 69%, 60% and 49% for SNPs with a MAF from 0.3% to 5% for 1M, 610k and 317k, respectively. None of the very rare variants （MAF 〈 0.3%） were well imputed. We conclude that the imputation accuracy of rare variants increases with higher density of genome-wide genotyping arrays when the size of the reference panel is small. Variants with lower MAF are more difficult to impute. These findings have important implications in the design and replication of large-scale sequencing studies.
基金This study was funded by Agri-Food and Agriculture Canada(Project AAFC J0000–75)
文摘The Genome Analysis Toolkit(GATK) is a popular set of programs for discovering and genotyping variants from next-generation sequencing data.The current GATK recommendation for RNA sequencing(RNA-seq) is to perform variant calling from individual samples,with the drawback that only variable positions are reported.Versions 3.0 and above of GATK offer the possibility of calling DNA variants on cohorts of samples using the HaplotypeCaller algorithm in Genomic Variant Call Format(GVCF) mode.Using this approach,variants are called individually on each sample,generating one GVCF file per sample that lists genotype likelihoods and their genome annotations.In a second step,variants are called from the GVCF files through a joint genotyping analysis.This strategy is more flexible and reduces computational challenges in comparison to the traditional joint discovery workflow.Using a GVCF workflow for mining SNP in RNA-seq data provides substantial advantages,including reporting homozygous genotypes for the reference allele as well as missing data.Taking advantage of RNA-seq data derived from primary macrophages isolated from 50 cows,the GATK joint genotyping method for calling variants on RNA-seq data was validated by comparing this approach to a so-called "per-sample" method.In addition,pair-wise comparisons of the two methods were performed to evaluate their respective sensitivity,precision and accuracy using DNA genotypes from a companion study including the same 50 cows genotyped using either genotyping-by-sequencing or with the Bovine SNP50 Beadchip(imputed to the Bovine high density).Results indicate that both approaches are very close in their capacity of detecting reference variants and that the joint genotyping method is more sensitive than the per-sample method.Given that the joint genotyping method is more flexible and technically easier,we recommend this approach for variant calling in RNA-seq experiments.
基金supported by the project "Transmission Mode of Tuberculosis" (2008ZX100/03-010-02)"Warning Mode of Tuberculosis" (2008ZX10003-008) of the National Key Programme of Mega Infectious Diseasesupported by the project "Mycobacterium tuberculosis genome SNP analysis and research on the origin of the Beijing family strains" (2011SKLID208) of State Key Laboratory for Infectious Disease Prevention and Control
文摘Objective To evaluate four candidate variable number tandem repeat （VNTR） loci for genotyping Mycobacterium tuberculosis complex strains. Methods Genomic sequences for two M. tuberculosis strains （CCDC5079 and CCDC5180） were generated, and using published sequence data, four candidate VNTR loci were identified. The VNTRs were used to genotype 225 Chinese clinical M. tuberculosis complex strains. The discriminatory power of the VNTRs was evaluated using BioNumerics 5.0 software. Results The Hunter-Gaston Index （HGI） for BJ1, BJ2, BJ3, and BJ4 loci was 0.634, 0.917, 0.697, and 0.910, respectively. Combining all four loci gave an HGI value of 0.995, thus confirming that the genotyping had good discriminatory power. The HGI values for BJ1, BJ2, BJ3, and BJ4, obtained from Beijing family strain genotyping, were 0.447, 0.878, 0.315, and 0.850, respectively. Combining all four loci produced an HGI value of 0.988 for genotyping the Beijing family strains. We observed unique patterns for M. boris and M. africanum strains from the four loci. Conclusion We have shown that the four VNTR loci can be successfully used for genotyping M. tuberculosis complex strains. Notably, these new loci may provide additional information about Chinese M. tuberculosis isolates than that currently afforded by established VNTR loci typing.
基金supported by Natural Science foundation(Grant No.31100105)China Mega-Project for Infectious Disease(2012ZX10004-215 and 2013ZX10004221)
文摘Objective Human Lyme Borreliosis （LB）, which is caused by Borrefia burgdorferi sensu lato （B. burgdorferi）, has been identified as a major arthropod-borne infectious disease in China. We aimed to develop a multiple locus variable-number tandem repeat （VNTR） analysis （MLVA） assay for the genotyping of Borrelia burgdorJ：eri strains detected in China. Methods B. garinii PBi complete 904.246 kb chromosome and two plasmids （cp26 and Ip54） were screened by using Tandem Repeats Finder program for getting potential VNTR loci, the potential VNTR loci were analyzed and identified with PCR and the VNTR loci data were analyzed and MLVA clustering tree were constrcted by using the categorical coefficient and the unweighted pair-group method with arithmetic means （UPGMA）. Results We identified 5 new VNTR loci through analyzing 47 potential VNTR loci. We used the MLVA protocol to analyse 101 B. burgdorferi strains detected in China and finally identified 51 unique genotypes in 4 major clusters including B. burgdorferi sensu stricto （B.b.s.s）, B. garinii, B. a[zelii, and B. valaisiana, consistent with the current MLSA phylogeny studies. The allele numbers of VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5 were 7, 3, 9, 7, and 6. The Hunter-Gaston index （HGI） of five VNTR loci were 0.79, 0.22, 0.77, 0.71, and 0.67, respectively. The combined HGI of five VNTR loci was 0.96. Clustering of the strains of Xinjiang, Inner Mongolia and Heilongjiang was confirmed, and this situation was consistent with the close geographical distribution of those provinces. Conclusion The MLVA protocol esytablished in this study is easy and can show strains＇ phylogenetic relationships to distinguish the strains of Borrelia species. It is useful for further phylogenetic and epidemiological analyses of Borrelia strains.
基金supported by the fundings from the Region Pays de la Loire through Biogenouest,the IBiSA Program,Fondation Progreffethe French Government through the "Investissement d'avenir" program "TEFOR" project,managed by the National Research Agency(No.ANR-II-INSB-0014)the context of the "Investissement d'avenir" program LabEX IGO of the IHU-CESTI projects managed by the National Research Agency(Nos.ANR-11-LABX-001601 and ANR-10-IBHU-005,respectively)
文摘The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout（KO） or knockin（KI） animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA（gDNA） followed by an automated capillary electrophoresis step which reveals a heteroduplexes（HD） signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.
基金financially supported by Fundamental Research Funds for the Central Universities of China(2572014EA06)National Natural Science Foundation of China(NSFC 31572285)Study on Resource Survey Technology for Tiger and Amur Leopard Population(State Forestry Administration)
文摘In modern wildlife ecological research,feces is the most common non-invasive source of DNA obtained in the field and polymerase chain reaction(PCR) technology based on microsatellite markers is used to mine genetic information contained within.This is especially the case for endangered species.However,there are risks associated with this genotyping method because of the poor quality of fecal DNA.In this study,we assessed genotyping risk across 12 microsatellite loci commonly used in previous tiger studies using blood and fecal DNA from captive Amur tigers(Panthera tigris altaica).To begin,we developed an index termed the accumulated matching rate of genotypes(R)between positive DNA(blood samples) and fecal DNA to explore the correct genotyping probability of a certain microsatellite locus.We found that different microsatelliteloci had different genotyping risks and required different PCR amplification protocols.The genotyping errors we detected altered population genetic parameters and potentially impact subsequent analyses.Based on these findings,we recommend that:(1) four loci(E7,Fca094,Pti007 and Pti010) of 12 loci are not suitable for Amur tiger genetic research because of a low Rand difficulty reaching a stable status;(2) the Rof the 12 microsatellite loci plateaued differently,and considering limited budgets,amplification times of some loci could be increased when using fecal samples; and(3) future genetic analysis of wild Amur tigers should be corrected by genotyping error rates(1-R).
基金supported by the National Natural Science Foundation of China(31871653 to K.L.,31830067 to J.L.)the Talent Project of Chongqing Natural Science Foundation(cstc2021ycjhbgzxm0033 to K.L.)Germplasm Creation Special Program of Southwest University.
文摘Rapeseed(Brassica napus)is an oil crop grown worldwide,making it a key plant species in molecular breeding research.However,the complexity of its polyploid genome increases sequencing costs and reduces sequencing accuracy.Target capture coupled with high-throughput sequencing is an efficient approach for detecting genetic variation at genomic regions or loci of interest.In this study,588 resequenced accessions of rapeseed were used to develop a target capture sequencing SNP genotyping platform named BnaPan50T.The platform comprised 54,765,with 54,058 resequenced markers from the pan-genome,and 855 variant trait-associated markers for 12 agronomic traits.The capture quality of BnaPan50T was demonstrated well in 12 typical accessions.Compared with a conventional genotyping array,BnaPan50T has a high SNP density and a high proportion of SNPs in unique physical positions and in annotated functional genes,promising wide application.Target capture sequencing and wholegenome resequencing in 90 doubled-haploid lines yielded 60%specificity,78%uniformity within tenfold coverage range,and 93%genotyping accuracy for the platform.BnaPan50T was used to construct a genetic map for quantitative trait loci(QTL)mapping,identify 21 unique QTL,and predict several candidate genes for yield-related traits in multiple environments.A set of 132 core SNP loci was selected from BnaPan50T to construct DNA fingerprints and germplasm identification resources.This study provides genomics resources to support target capture sequencing,genetic analysis and genomic breeding of rapeseed.
文摘To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS： Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 （33%）, 41 （33%） and 45 （36%） samples, and genotype C in 76 （60%）, 76 （60%） and 81 （64%） samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real- time PCR was the rapidest and cheapest among the three assays. CONCLUSION： Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.
基金This study was funded by the Genomic Selection in Animals and Plants(GenSAP)research project financed by the Danish Council of Strategic Research(Aarhus,Denmark).Xiao Wang received Ph.D.stipends from the Technical University of Denmark(DTU Bioinformatics and DTU Compute),Denmark,and the China Scholarship Council,China.
文摘Background:Genotyping by sequencing(GBS)still has problems with missing genotypes.Imputation is important for using GBS for genomic predictions,especially for low depths,due to the large number of missing genotypes.Minor allele frequency(MAF)is widely used as a marker data editing criteria for genomic predictions.In this study,three imputation methods(Beagle,IMPUTE2 and FImpute software)based on four MAF editing criteria were investigated with regard to imputation accuracy of missing genotypes and accuracy of genomic predictions,based on simulated data of livestock population.Results:Four MAFs(no MAF limit,MAF≥0.001,MAF≥0.01 and MAF≥0.03)were used for editing marker data before imputation.Beagle,IMPUTE2 and FImpute software were applied to impute the original GBS.Additionally,IMPUTE2 also imputed the expected genotype dosage after genotype correction(GcIM).The reliability of genomic predictions was calculated using GBS and imputed GBS data.The results showed that imputation accuracies were the same for the three imputation methods,except for the data of sequencing read depth(depth)=2,where FImpute had a slightly lower imputation accuracy than Beagle and IMPUTE2.GcIM was observed to be the best for all of the imputations at depth=4,5 and 10,but the worst for depth=2.For genomic prediction,retaining more SNPs with no MAF limit resulted in higher reliability.As the depth increased to 10,the prediction reliabilities approached those using true genotypes in the GBS loci.Beagle and IMPUTE2 had the largest increases in prediction reliability of 5 percentage points,and FImpute gained 3 percentage points at depth=2.The best prediction was observed at depth=4,5 and 10 using GcIM,but the worst prediction was also observed using GcIM at depth=2.Conclusions:The current study showed that imputation accuracies were relatively low for GBS with low depths and high for GBS with high depths.Imputation resulted in larger gains in the reliability of genomic predictions for GBS with lower depths.These results suggest that the application of IMPUTE2,based on a corrected GBS(GcIM)to improve genomic predictions for higher depths,and FImpute software could be a good alternative for routine imputation.
