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1.
An Acad Bras Cienc ; 91(suppl 3): e20190339, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31460595

RESUMO

Genetic drift is the fortuitous occurrence of genetic events that when they become fixed modify the genome of populations. They can take the form of mutations of single nucleotides (SNPs), the insertion or deletion of short sequences (Indels) or the repetitions of short sequences (CNV i.e. copy number variants) or long insertions or deletion (structural modifications). Their frequency is 10-9 to 10-8 depending on the species, or 50 to 100 per birth in humans. The incidence of these de novo mutations is higher when the father is old at conception. It thus appears that genetic drift, which constitutes the initial element of evolution, has a very strong dynamics. Its intervention in the appearance or disappearance of some major phenotypes is complicated by the uncertainties about the genetic mechanisms in heritability which, paradoxically, are only partially understood.


Assuntos
Evolução Molecular , Deriva Genética , Mutação INDEL/genética , Mamíferos/genética , Animais , Humanos , Camundongos , Ratos
2.
Gene ; 712: 143965, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31279708

RESUMO

Recently, we read the published article in GENE. Dong et al. presented the evaluation of the MDM2 40-bp insertion/deletion status in Hepatocellular carcinoma patients (Dong et al., 2012). The authors stated that the insertion allele showed a 521-bp band and the deletion allele showed a 481-bp band on agarose gel electrophoresis. While it seems that these reported sizes for insertion and deletion alleles of MDM2 are incorrect. Our analysis using the primers indicated that the length of insertion and deletion fragments will be 481 and 441 bps, respectively. Actually, 40-bp is added to the fragment length instead of reducing the 40-bp. In the 'UCSC In-Silico PCR' tool, the length of the amplified fragment using mentioned primers is 481-bp including the sequence of 40-bp insertion allele (5'-(A)5GCTGCA(GAAGG)2ATATAACTTTAT(A)7-3') (Reference SNP (rs) Report, n.d.). Therefore, the fragment including the deletion allele will be 441-bp.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Alelos , Predisposição Genética para Doença , Humanos , Mutação INDEL , Polimorfismo Genético , Proteínas Proto-Oncogênicas c-mdm2/genética
3.
Med Oncol ; 36(8): 71, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270633

RESUMO

BRCA1 is involved in double-strand DNA damage repair pathways, and mutations in the gene are associated with hereditary breast and ovarian cancers. With great help of the development of high-throughput DNA sequencing techniques numerous single-nucleotide polymorphisms (SNPs) and insertion deletion (Indel) mutations are detected on both coding and non-coding/regulatory regions of the BRCA1. Mutations may cause pathogenic or benign changes on the protein function or affect its expression. In the last decade, use of genetic screening tests to detect mutations on such genes has become greatly popular. However, it is very important to know the effect of the detected mutations, which is mostly possible by the use of predictive softwares, and also the related family history to be able to correctly analyse the screening results and to inform the patient. Therefore, use of in silico and in vitro techniques to score the pathogenicity of detected variants on genes like BRCA1 is now of great importance. Otherwise, results obtained from screening tests and family history cannot be analysed precisely.


Assuntos
Proteína BRCA1/genética , Genes BRCA1 , Testes Genéticos/métodos , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Neoplasias da Mama/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Neoplasias Ovarianas/genética , Polimorfismo de Nucleotídeo Único
4.
BMC Genomics ; 20(1): 459, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170919

RESUMO

BACKGROUND: The most widely used human genome reference assembly hg19 harbors minor alleles at 2.18 million positions as revealed by 1000 Genome Phase 3 dataset. Although this is less than 2% of the 89 million variants reported, it has been shown that the minor alleles can result in 30% false positives in individual genomes, thus misleading and burdening downstream interpretation. More alarming is the fact that, significant percentage of variants that are homozygous recessive for these minor alleles, with potential disease implications, are masked from reporting. RESULTS: We have demonstrated that the false positives (FP) and false negatives (FN) can be corrected for by simply replacing nucleotides at the minor allele positions in hg19 with corresponding major allele. Here, we have effectively replaced 2.18 million minor alleles Single Nucleotide Polymorphism (SNPs), Insertion and Deletions (INDELs), Multiple Nucleotide Polymorphism (MNPs) in hg19 with the corresponding major alleles to create an ethnically normalized reference genome called hg19KIndel. In doing so, hg19KIndel has both corrected for sequencing errors acknowledged to be present in hg19 and has improved read alignment near the minor alleles in hg19. CONCLUSION: We have created and made available a new version human reference genome called hg19KIndel. It has been shown that variant calling using hg19KIndel, significantly reduces false positives calls, which in-turn reduces the burden from downstream analysis and validation. It also improved false negative variants call, which means that the variants which were getting missed due to the presence of minor alleles in hg19, will now be called using hg19KIndel. Using hg19KIndel, one even gets a better mapping percentage when compared to currently available human reference genome. hg19KIndel reference genome and its auxiliary datasets are available at https://doi.org/10.5281/zenodo.2638113.


Assuntos
Grupos Étnicos/genética , Variação Genética , Genoma Humano , Alelos , Bases de Dados de Ácidos Nucleicos , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Padrões de Referência , Análise de Sequência de DNA
5.
Croat Med J ; 60(3): 246-249, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31187952

RESUMO

The diagnosis of cystic fibrosis (CF) is commonly confirmed by molecular genetics with the presence of specific mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report a case of cystic fibrosis (CF) in a 15-year-old female patient who is a compound heterozygote for CFTR gene, with delta F508 and Tyr109Glyfs mutations detected. This is the first detailed description of such a case in the medical literature. The primary CF presentation occurred at the age of 9 in the form of gastrointestinal symptoms including greasy, bulky, and foul-smelling stool. The patient exhibited delayed growth, with her height and weight being below the 5th centile for age according to the World Health Organization growth curves. Pancreatic enzyme supplement treatment was started immediately, alongside high-fat and high-calorie diet, resulting in patient's recovery and development. DNA analysis of CFTR gene demonstrated the presence of del. F508 mutation and a rare combining deletion and insertion mutation p. Tyr109Glyfs. The combination of the two mutations is very rare in CF patients and is therefore valuable to document this case in order to provide information on disease progression, therapy options, and outcomes. With standard treatment and early diagnosis, the patient is currently doing well and is not restricted by the disease in her daily and sports activities.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/terapia , Adolescente , Criança , Fibrose Cística/diagnóstico , Feminino , Heterozigoto , Humanos , Mutação INDEL
6.
BMC Bioinformatics ; 20(1): 342, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208315

RESUMO

BACKGROUND: Whole exome sequencing (WES) is a cost-effective method that identifies clinical variants but it demands accurate variant caller tools. Currently available tools have variable accuracy in predicting specific clinical variants. But it may be possible to find the best combination of aligner-variant caller tools for detecting accurate single nucleotide variants (SNVs) and small insertion and deletion (InDels) separately. Moreover, many important aspects of InDel detection are overlooked while comparing the performance of tools, particularly its base pair length. RESULTS: We assessed the performance of variant calling pipelines using the combinations of four variant callers and five aligners on human NA12878 and simulated exome data. We used high confidence variant calls from Genome in a Bottle (GiaB) consortium for validation, and GRCh37 and GRCh38 as the human reference genome. Based on the performance metrics, both BWA and Novoalign aligners performed better with DeepVariant and SAMtools callers for detecting SNVs, and with DeepVariant and GATK for InDels. Furthermore, we obtained similar results on human NA24385 and NA24631 exome data from GiaB. CONCLUSION: In this study, DeepVariant with BWA and Novoalign performed best for detecting accurate SNVs and InDels. The accuracy of variant calling was improved by merging the top performing pipelines. The results of our study provide useful recommendations for analysis of WES data in clinical genomics.


Assuntos
Simulação por Computador , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento Completo do Exoma , Pareamento de Bases/genética , Exoma/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Curva ROC
7.
Forensic Sci Int ; 301: 382-387, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31220685

RESUMO

Sudden cardiac death (SCD) is referred to as sudden and unexpected death caused by cardiovascular diseases, in which a person preexisted heart disease or not. Compelling evidence indicates that SCD etiology have been predominantly affected by host genetic factors. However, how genetic variants play roles in the inherited risk component of SCD are still largely unknown. It has been reported that Desmoglein-2 (DSG2) mutations might be related to sudden death. In the present study, we used a candidate gene approach to investigate the associations between rs397729601 (a 2-base pair indel polymorphism) mapping to the 3'UTR of DSG2 with the risk of SCD. It is shown by logistic regression analysis that the risk of SCD has been significantly increased by the deletion allele of rs397729601 [odds ratio (OR)=1.51; 95% confidence interval (CI)=1.12-2.05; P=0.00559]. Additional genotype-phenotype analysis was performed to evaluate the mRNA level, revealing that human myocardium tissues with the deletion allele showed higher expression of DSG2. Dual luciferase activity analysis was conducted in an in vitro reporter gene system, suggesting that DSG2 expression could be regulated by rs397729601 which interrupted the binding of miR-933-3p with DSG2. We concluded that rs397729601 may affect the expression of DSG2 through miR-933-3p regulation, contributing to SCD susceptibility. Thus, rs397729601 may be used as a potential marker for molecular diagnosis and genetic counseling of SCD. Our findings need to be validated through replication and further functional studies.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Morte Súbita Cardíaca/etiologia , Desmogleína 2/genética , Mutação INDEL , Polimorfismo Genético , Estudos de Casos e Controles , China , Desmogleína 2/metabolismo , Feminino , Genética Forense , Predisposição Genética para Doença , Genótipo , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Medicine (Baltimore) ; 98(24): e15846, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31192914

RESUMO

Ischemic heart disease (IHD) has a genetic predisposition and a number of cardiovascular risk factors are known to be affected by genetic factors. Development of metabolic syndrome and insulin resistance, strongly influenced by lifestyle and environmental factors, frequently occur in subjects with a genetic susceptibility. The definition of genetic factors influencing disease susceptibility would allow to identify individuals at higher risk and thus needing to be closely monitored.To this end, we focused on a complex of soluble-N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), playing an important role in metabolic syndrome and insulin resistance, involved in endothelial dysfunction and heart disease. We assessed if genetic variants of the SNARE genes are associated with IHD.SNAP25 rs363050, Stx-1A rs4717806, rs2293489, and VAMP2 26bp ins/del genetic polymorphisms were analyzed in a cohort of 100 participants who underwent heart surgery; 56 of them were affected by IHD, while 44 were not. A statistical association of plasma glycemia and insulin resistance, calculated as Triglyceride glucose (TyG) index, was observed in IHD (P < .001 and P = .03, respectively) after binomial logistic stepwise regression analysis, adjusted by age, gender, diabetes positivity, waist circumference, and cholesterol plasma level. Among genetic polymorphisms, rs4717806(A) and rs2293489(T), as well as the rs4717806 - rs2293489 (A-T) haplotype were associated with higher risk for IHD (Pc = .02; Pc = .02; P = .04, respectively). Finally, a statistical association of rs4717806(AA) genotype with higher TyG index in IHD patients (P = .03) was highlighted by multiple regression analysis considering log-transformed biochemical parameters as dependent variable and presence of coronary artery disease, age, gender, waist circumference, presence of diabetes as predictors. These results point to a role of the Stx-1A rs4717806 SNP in IHD, possibly due to its influence on Stx-1A expression and, as a consequence, on insulin secretion and glucose metabolism.


Assuntos
Estudos de Associação Genética/métodos , Isquemia Miocárdica/genética , Isquemia Miocárdica/cirurgia , Polimorfismo de Nucleotídeo Único , Sintaxina 1/genética , Idoso , Idoso de 80 Anos ou mais , Procedimentos Cirúrgicos Cardíacos , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Humanos , Mutação INDEL , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteína 25 Associada a Sinaptossoma/genética , Proteína 2 Associada à Membrana da Vesícula/genética
9.
Int J Syst Evol Microbiol ; 69(7): 2057-2063, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31091185

RESUMO

In the last 4 years, most of the species previously classified as members of the genus Burkholderia have been transferred to the novel genera Paraburkholderia, Caballeronia, Robbsia, Mycetohabitans and Trinickia. However, there have been objections to splitting the genus Burkholderiasensu lato, and based on this taxonomic opinion, strain RPE64T, which has the 16S rRNA gene sequence identical to that of Caballeronia peredens LMG 29314T, has recently been proposed as the type strain of Burkholderia insecticolasp. nov. The arguments against the split were analysed in this study and found to be not convincing enough to revise the taxonomic positions of members of the novel genera. Therefore, based on the results of phylogenetic analyses, including comparisons of 16S rRNA gene sequences and those of concatenated proteins, as well as on the fact that strain RPE64T had all molecular signatures included as Caballeronia-specific markers in the genus description, we propose to reclassify B. insecticola as Caballeronia insecticola comb. nov. The results of this study also showed that 'Burkholderia novacaledonica' and 'Burkholderia ultramafica' should be transferred to the genera Caballeronia and Paraburkholderia, respectively.


Assuntos
Burkholderia/classificação , Burkholderiaceae/classificação , Filogenia , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Mutação INDEL , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA
10.
BMC Genomics ; 20(1): 323, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035925

RESUMO

BACKGROUND: Artificial induction of mutagenesis is effective for genetic resource innovation and breeding. However, the traditional mutation methods for fish breeding are not convenient or safe for daily use. Hence, development of a simple, safe and effective mutagenesis method with a high mutation rate and applicability to multiple fish species, is needed. RESULTS: We reported the first successful mutagenesis in a marine aquaculture fish species, Japanese flounder, Paralichthys olivaceus, using a novel atmosphere and room temperature plasma (ARTP) mutagenesis tool. ARTP treatment time was optimized for the fertilized eggs and sperm, respectively. Eggs fertilized for 60 min were treated by ARTP with a radio-frequency power input of 120 W, and the ARTP treatment time was 25 min. Under an ARTP radio-frequency power input of 200 W, the optimal treatment time for sperm diluted with Ringer's solution by 1:40 v/v was 10 min. The ARTP-treated group presented differences in morphological traits such as body height, total length among individuals at day 90 after hatching. Whole-genome sequencing was used to reveal the mutation features of ARTP-treated individuals collected at day 120 after hatching. In total, 69.25Gb clean data were obtained from three controls and eight randomly selected ARTP-treated individuals, revealing 240,722 to 322,978 SNPs and 82,149 to 86,798 InDels located in 17,394~18,457 and 12,907~13,333 genes, respectively. The average mutation rate reached 0.064% at the genome level. Gene ontology clustering indicated that genes associated with cell components, binding function, catalytic activity, cellular process, metabolic process and biological regulation processes had higher mutation rates. CONCLUSIONS: ARTP mutagenesis is a useful method for breeding of fish species to accelerate the selection of economically important traits that would benefit the aquaculture industry, given the variety of mutations detected.


Assuntos
Linguado/genética , Gases em Plasma , Ondas de Rádio , Animais , Tamanho Corporal , Cruzamento , Análise por Conglomerados , Linguado/crescimento & desenvolvimento , Mutação INDEL , Japão , Masculino , Mutagênese , Polimorfismo de Nucleotídeo Único , Espermatozoides/efeitos da radiação , Temperatura Ambiente , Sequenciamento Completo do Genoma , Zigoto/efeitos da radiação
11.
Forensic Sci Int Genet ; 41: 107-119, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31071519

RESUMO

The deconvolution of DNA mixtures has gathered the attention of forensic DNA scientists for over two decades. To enhance mixture deconvolution capabilities, a new generation of sensitive DNA-typing approaches has been recently proposed. In this review, we describe novel, forensically relevant multi-SNP loci (i.e., microhaplotypes or microhaps), compound markers (i.e., DIP-STRs, SNP-STRs and DIP-SNPs) and lineage markers (i.e., rapidly mutating Y chromosome STRs) that improve the deconvolution of two and more than two-person mixtures typed using conventional STR, binary and non-binary loci. We explore the features and applications of these emerging molecular biomarkers with respect to their ability to forensically detect same-or-opposite sex donors. Finally, we discuss the impact of initial massively parallel sequencing (MPS) investigations of STR, microhaplotype and SNP/indel assays for DNA mixture profiling.


Assuntos
Impressões Digitais de DNA/métodos , DNA/genética , Marcadores Genéticos , Cromossomos Humanos Y , Eletroforese Capilar , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
12.
Forensic Sci Int Genet ; 41: 128-136, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31079022

RESUMO

In addition to commonly used short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), insertion and deletion polymorphisms (InDels) have considerable potential in the field of forensic genetics because they combine desirable characteristics of both STRs and SNPs. In the present study, the SifaInDel 45plex system was designed to amplify 45 InDel markers, including 27 autosomal InDels (A-InDels), 16 X chromosome InDels (X-InDels) and two Y chromosome InDels (Y-InDels), simultaneously in a single PCR procedure and then detect products by capillary electrophoresis (CE). We also optimized the PCR conditions for the novel panel and performed several validation studies including repeatability/reproducibility, concordance, accuracy, sensitivity, stability, species specificity and population genetics. The results confirmed that full profiles could be obtained from ≥62.5 pg of input DNA and from a series of challenging samples encountered in routine casework. The SifaInDel 45plex panel could tolerate different concentrations of inhibitors, such as ≤50 µM hematin, ≤20 ng/µL nigrosine and ≤8000 ng/µL urea. In a population investigation, for the 27 A-InDels, the combined power of discrimination (CPD) exceeded 0.999999, and the combined power of exclusion in duos (CPED) and trios (CPET) was 0.955118 and 0.997754, respectively. For the 16 X-InDels, the combined PDMale and PDFemale was computed as 0.999845 and 0.999998, respectively, and the combined mean exclusion chance in father/daughter or mother/son duos (MECDuo) and mean exclusion chance in standard trios involving daughters (MECTrio) was 0.976220 and 0.998163, respectively. In addition, the two Y-InDels could play a role in correctly determining gender. Overall, the established SifaInDel 45plex panel is a well-performing, reliable and robust multiplex system that stands out for combining a considerable number of A-indels, X-indels and Y-indels based on a CE platform. The population study results also demonstrated that the SifaInDel 45plex panel could be a valid complementary approach for human identification and complex kinship analysis.


Assuntos
Cromossomos Humanos X , Cromossomos Humanos Y , Genética Forense , Marcadores Genéticos , Mutação INDEL , Polimorfismo Genético , Animais , Eletroforese Capilar , Feminino , Frequência do Gene , Genética Populacional , Genótipo , Humanos , Masculino , Reação em Cadeia da Polimerase , Especificidade da Espécie
13.
Forensic Sci Int Genet ; 41: 159-167, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31136932

RESUMO

At present, several mature ancestry informative SNP (AISNP) panels are used to distinguish between continental regions of the world, but a more accurate division within the continent requires a secondary panel to complete. However, many AISNPs for the subgroup ancestry inference are selected from the Kidd Lab panel of 55 AISNPs or other published papers. These panels inevitably lack valuable markers for subgroup ancestry inference. Therefore, instead of choosing from the published panels, we used the 1000 Genomes Project to screen potentially informational markers in Asian populations, including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDels). The allele frequencies of all autosomal SNPs and InDels of the 1000 Genomes Project were compared between 10 populations in Asia to identify markers with the largest pairwise allele frequency differences. Finally, we established a second-tier panel of 18 AIMs in this study, which not only divided the 26 populations of the 1000 Genomes Project into six clusters, but also divided the Asia subgroup into four clusters: Gujarati, East Asia, Southeast Asia and South Asia.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Marcadores Genéticos , Genética Populacional , Ásia , Frequência do Gene , Genótipo , Humanos , Mutação INDEL , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
14.
Med Sci Monit ; 25: 3390-3396, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31064975

RESUMO

BACKGROUND This study aimed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion polymorphisms with left ventricular hypertrophy (LVH) in Han and Uighur hypertension-OSAHS (obstructive sleep apnea hypopnea syndrome) patients in China. MATERIAL AND METHODS A total of 162 Han and 72 Uygur patients with hypertension-OSAHS were independently subdivided into an LVH group and a non-LVH (NLVH) group based on the left ventricular mass index. The insertion/deletion polymorphisms of ACE gene were determined by polymerase chain reaction. The association of ACE gene insertion/deletion polymorphisms with LVH was assessed by chi-squared test. Logistic regression analysis was performed to obtain the odds ratios and 95% confidence intervals for the risk of LVH after adjusting for confounding factors. RESULTS In Uighur patients, the distributions of D allele and DD genotype showed significant differences between the LVH group and the NLVH group. The difference of DD genotype remained significant after multivariate adjustment. In contrast, no significant differences were observed in the distributions of D allele and DD genotype between the LVH group and the NLVH group in Han patients. Moreover, moderate-severe OSAHS was an independent risk factor for LVH. CONCLUSIONS D allele and DD genotype of ACE gene are possible genetic markers for the risk of LVH in Uighur but not Han hypertension-OSAHS patients.


Assuntos
Hipertrofia Ventricular Esquerda/genética , Peptidil Dipeptidase A/genética , Apneia Obstrutiva do Sono/genética , Adulto , Alelos , Grupo com Ancestrais do Continente Asiático/genética , China , Grupos Étnicos/genética , Feminino , Frequência do Gene/genética , Genótipo , Humanos , Hipertensão/complicações , Hipertensão/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Mutação INDEL/genética , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/fisiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Fatores de Risco , Apneia Obstrutiva do Sono/complicações
15.
Sci Data ; 6(1): 12, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931948

RESUMO

In barley and other cereal crops, phenological diversity drives adaptation to different cultivation areas. Improvement of barley yield and quality traits requires adaptation to specific production areas with introgression of favorable alleles dependent upon precise identification of the underlying genes. Combining targeted sequence capture systems with next-generation sequencing provides an efficient approach to explore target genetic regions at high resolution, and allows rapid discovery of thousands of genetic polymorphisms. Here, we apply a versatile target-capture method to detect genome-wide polymorphisms in 174 flowering time-related genes, chosen based on prior knowledge from barley, rice, and Arabidopsis thaliana. Sequences were generated across a phenologically diverse panel of 895 barley varieties, resulting a high mean depth coverage of ~25x allowing reliable discovery and calling of insertion-deletion (InDel) and single nucleotide polymorphisms (SNPs). Sequences of InDel and SNPs from the targeted enrichment were utilized to develop 67 Kompetitive Allele Specific PCR (KASP) markers for validation. This work provides researchers and breeders a comprehensive molecular toolkit for the selection of phenology-related traits in barley.


Assuntos
Genoma de Planta , Hordeum/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
16.
BMC Bioinformatics ; 20(1): 213, 2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31029080

RESUMO

BACKGROUND: Next Generation Sequencing (NGS) is a commonly used technology for studying the genetic basis of biological processes and it underpins the aspirations of precision medicine. However, there are significant challenges when dealing with NGS data. Firstly, a huge number of bioinformatics tools for a wide range of uses exist, therefore it is challenging to design an analysis pipeline. Secondly, NGS analysis is computationally intensive, requiring expensive infrastructure, and many medical and research centres do not have adequate high performance computing facilities and cloud computing is not always an option due to privacy and ownership issues. Finally, the interpretation of the results is not trivial and most available pipelines lack the utilities to favour this crucial step. RESULTS: We have therefore developed a fast and efficient bioinformatics pipeline that allows for the analysis of DNA sequencing data, while requiring little computational effort and memory usage. DNAscan can analyse a whole exome sequencing sample in 1 h and a 40x whole genome sequencing sample in 13 h, on a midrange computer. The pipeline can look for single nucleotide variants, small indels, structural variants, repeat expansions and viral genetic material (or any other organism). Its results are annotated using a customisable variety of databases and are available for an on-the-fly visualisation with a local deployment of the gene.iobio platform. DNAscan is implemented in Python. Its code and documentation are available on GitHub: https://github.com/KHP-Informatics/DNAscan . Instructions for an easy and fast deployment with Docker and Singularity are also provided on GitHub. CONCLUSIONS: DNAscan is an extremely fast and computationally efficient pipeline for analysis, visualization and interpretation of NGS data. It is designed to provide a powerful and easy-to-use tool for applications in biomedical research and diagnostic medicine, at minimal computational cost. Its comprehensive approach will maximise the potential audience of users, bringing such analyses within the reach of non-specialist laboratories, and those from centres with limited funding available.


Assuntos
Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Interface Usuário-Computador , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Bases de Dados Factuais , HIV-1/genética , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Sequenciamento Completo do Genoma
17.
BMC Cancer ; 19(1): 396, 2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31029168

RESUMO

BACKGROUND: With the introduction of Olaparib treatment for BRCA-deficient recurrent ovarian cancer, testing for somatic and/or germline mutations in BRCA1/2 genes in tumor tissues became essential for treatment decisions. In most cases only formalin-fixed paraffin-embedded (FFPE) samples, containing fragmented and chemically modified DNA of minor quality, are available. Thus, multiplex PCR-based sequencing is most commonly applied in routine molecular testing, which is predominantly focused on the identification of known hot spot mutations in oncogenes. METHODS: We compared the overall performance of an adjusted targeted capture-based enrichment protocol and a multiplex PCR-based approach for calling of pathogenic SNVs and InDels using DNA extracted from 13 FFPE tissue samples. We further applied both strategies to seven blood samples and five matched FFPE tumor tissues of patients with known germline exon-spanning deletions and gene-wide duplications in BRCA1/2 to evaluate CNV detection based solely on panel NGS data. Finally, we analyzed DNA from FFPE tissues of 11 index patients from families suspected of having hereditary breast and ovarian cancer, of whom no blood samples were available for testing, in order to identify underlying pathogenic germline BRCA1/2 mutations. RESULTS: The multiplex PCR-based protocol produced inhomogeneous coverage among targets of each sample and between samples as well as sporadic amplicon drop out, leading to insufficiently or non-covered nucleotides, which subsequently hindered variant detection. This protocol further led to detection of PCR-artifacts that could easily have been misinterpreted as pathogenic mutations. No such limitations were observed by application of an adjusted targeted capture-based protocol, which allowed for CNV calling with 86% sensitivity and 100% specificity. All pathogenic CNVs were confirmed in the five matched FFPE tumor samples from patients carrying known pathogenic germline mutations and we additionally identified somatic loss of the second allele in BRCA1/2. Furthermore we detected pathogenic BRCA1/2 variants in four the eleven FFPE samples from patients of whom no blood was available for analysis. CONCLUSIONS: We demonstrate that an adjusted targeted capture-based enrichment protocol is superior to commonly applied multiplex PCR-based protocols for reliable BRCA1/2 variant detection, including CNV-detection, using FFPE tumor samples.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Ovarianas/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Variações do Número de Cópias de DNA/genética , Feminino , Formaldeído/química , Humanos , Mutação INDEL , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/diagnóstico , Inclusão em Parafina , Linhagem , Reprodutibilidade dos Testes , Fixação de Tecidos
18.
Anim Genet ; 50(3): 279-282, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974000

RESUMO

Glutaminyl-peptide cyclotransferase-like (QPCTL) is an isoenzyme of glutaminyl-peptide cyclotransferase (QPCT). QPCTL and QPCT catalyze the formation of N-terminal modified pyroglutamate-fractalkine and the chemokine CCL2. The objective of this study was to investigate the association between insertions/deletions in the chicken QPCTL promoter region with growth traits in chickens. We first detected two insertion/deletion variants of QPCTL via whole-genome resequencing analysis of DNA samples from Xichuan chickens. A total of 1896 individuals from 12 breeds were genotyped for 52- and 224-bp insertions/deletions. We found two novel insertions/deletions in the promoter region of the chicken QPCTL gene and studied their association with chicken body weight and carcass traits. Our findings show that QPCTL can be a molecular marker for chicken genetics and breeding programs.


Assuntos
Aminoaciltransferases/genética , Galinhas/genética , Mutação INDEL , Regiões Promotoras Genéticas , Animais , Proteínas Aviárias/genética , Galinhas/classificação
19.
BMC Genomics ; 20(1): 304, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31014236

RESUMO

BACKGROUND: Although rapeseed (Brassica napus L.) mutant forming multiple siliques was morphologically described and considered to increase the silique number per plant, an important agronomic trait in this crop, the molecular mechanism underlying this beneficial trait remains unclear. Here, we combined bulked-segregant analysis (BSA) and whole genome re-sequencing (WGR) to map the genomic regions responsible for the multi-silique trait using two pools of DNA from the near-isogenic lines (NILs) zws-ms (multi-silique) and zws-217 (single-silique). We used the Euclidean Distance (ED) to identify genomic regions associated with this trait based on both SNPs and InDels. We also conducted transcriptome sequencing to identify differentially expressed genes (DEGs) between zws-ms and zws-217. RESULTS: Genetic analysis using the ED algorithm identified three SNP- and two InDel-associated regions for the multi-silique trait. Two highly overlapped parts of the SNP- and InDel-associated regions were identified as important intersecting regions, which are located on chromosomes A09 and C08, respectively, including 2044 genes in 10.20-MB length totally. Transcriptome sequencing revealed 129 DEGs between zws-ms and zws-217 in buds, including 39 DEGs located in the two abovementioned associated regions. We identified candidate genes involved in multi-silique formation in rapeseed based on the results of functional annotation. CONCLUSIONS: This study identified the genomic regions and candidate genes related to the multi-silique trait in rapeseed.


Assuntos
Brassica napus/genética , Genômica , Locos de Características Quantitativas/genética , Perfilação da Expressão Gênica , Mutação INDEL , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Homologia de Sequência do Ácido Nucleico
20.
Theor Appl Genet ; 132(7): 2125-2135, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31020387

RESUMO

KEY MESSAGE: Tomato male sterile-1526 locus was fine-mapped to an interval of 44.6 kb, and a B-class MADS-box gene TM6 was identified as the candidate gene. Male sterile lines have been widely used for hybrid seed production in many crop plants. The tomato male sterile-1526 (ms-1526) mutant displays abnormal stamens and exerted stigmas and is suitable for practical use. In this study, the ms-1526 locus was fine-mapped to a 44.6 kb interval that contained four putative genes. Thereinto, Solyc02g084630 encodes tomato B-class MADS-box gene TM6 (syn. TDR6), which plays an important role in stamen development. Sequencing revealed that there was a 12.7 kb deletion in the ms-1526 region, where the promoter and first four exons of the TM6 gene were absent. ms-1547, an allele of ms-1526, also contained the same deletion in the TM6 gene. And the other allele ms-15 mutant contained a single-nucleotide polymorphism (SNP, C to A) in the coding region of the TM6 gene, which led to a missense mutation (G to W). The codominant insertion/deletion (InDel) marker MS26D and codominant derived cleaved amplified polymorphic sequence (dCAPS) marker MS15C were developed based on the deletion and SNP, respectively. A real-time quantitative reverse-transcription PCR showed that expression of the TM6 gene was barely detectable in the flowers of the ms-1526 and ms-1547 mutants. In addition, other floral organ identity genes, pollen development marker genes, and pistil marker genes were differentially expressed between wild type and mutant flowers. These findings may facilitate functional analysis of the TM6 gene and help in the marker-assisted selection of ms-15 and its alleles in tomato breeding.


Assuntos
Flores/fisiologia , Lycopersicon esculentum/genética , Proteínas de Domínio MADS/genética , Infertilidade das Plantas/genética , Proteínas de Plantas/genética , Alelos , Mapeamento Cromossômico , Flores/genética , Marcadores Genéticos , Genótipo , Mutação INDEL , Lycopersicon esculentum/fisiologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Deleção de Sequência
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