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1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(5): 737-742, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31762247

RESUMO

OBJECTIVE: To establish SNaPShot-fluorescence capillary analysis (SNaPShot-FCA) assay for rapid detection of the genotype of aldehyde dehydrogenase 2 gene (ALDH2) rs671 locus. METHODS: The genomic DNA was extracted from peripheral blood cells. Using R6G-ddATP and cy5-ddGTP as fluorescent substrates, the ALDH2 gene was amplified by SNaPShot to generate DNA products with different fluorescent dyes at the 3' end. FCA was used to detect the products separated by agarose gel electrophoresis and recovered by gel recovery kit, and the genetype of ALDH2 polymorphism was analyzed by fluorescence spectrum. The samples were tested three times repeatedly and compared with the results of DNA sequencing. RESULTS: The optimal concentrations of R6G-ddATP and cy5-ddGTP were 1.4 µmol/L and 8.0 µmol/L, respectively. 106 samples were tested for ALDH2 genotype by SNaPShot-FCA under optimal conditions, including 67 of wild type (GG), 38 of hybrid type (AG), and 1 of mutant type (AA), which were consistent with the sequencing results. CONCLUSION: This study successfully established the SNaPShot-FCA for the micro-detection of ALDH2 genotype for the rapid screening and identification of ALDH2 gene.


Assuntos
Aldeído-Desidrogenase Mitocondrial/genética , Genótipo , Análise de Sequência de DNA/métodos , Fluorescência , Humanos
2.
BMC Bioinformatics ; 20(1): 498, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615395

RESUMO

BACKGROUND: Selecting the proper parameter settings for bioinformatic software tools is challenging. Not only will each parameter have an individual effect on the outcome, but there are also potential interaction effects between parameters. Both of these effects may be difficult to predict. To make the situation even more complex, multiple tools may be run in a sequential pipeline where the final output depends on the parameter configuration for each tool in the pipeline. Because of the complexity and difficulty of predicting outcomes, in practice parameters are often left at default settings or set based on personal or peer experience obtained in a trial and error fashion. To allow for the reliable and efficient selection of parameters for bioinformatic pipelines, a systematic approach is needed. RESULTS: We present doepipeline, a novel approach to optimizing bioinformatic software parameters, based on core concepts of the Design of Experiments methodology and recent advances in subset designs. Optimal parameter settings are first approximated in a screening phase using a subset design that efficiently spans the entire search space, then optimized in the subsequent phase using response surface designs and OLS modeling. Doepipeline was used to optimize parameters in four use cases; 1) de-novo assembly, 2) scaffolding of a fragmented genome assembly, 3) k-mer taxonomic classification of Oxford Nanopore Technologies MinION reads, and 4) genetic variant calling. In all four cases, doepipeline found parameter settings that produced a better outcome with respect to the characteristic measured when compared to using default values. Our approach is implemented and available in the Python package doepipeline. CONCLUSIONS: Our proposed methodology provides a systematic and robust framework for optimizing software parameter settings, in contrast to labor- and time-intensive manual parameter tweaking. Implementation in doepipeline makes our methodology accessible and user-friendly, and allows for automatic optimization of tools in a wide range of cases. The source code of doepipeline is available at https://github.com/clicumu/doepipeline and it can be installed through conda-forge.


Assuntos
Genômica/métodos , Análise de Sequência de DNA/métodos , Software , Francisella tularensis/genética , Genoma Bacteriano , Nanoporos
3.
BMC Bioinformatics ; 20(1): 496, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615419

RESUMO

BACKGROUND: When applying genomic medicine to a rare disease patient, the primary goal is to identify one or more genomic variants that may explain the patient's phenotypes. Typically, this is done through annotation, filtering, and then prioritization of variants for manual curation. However, prioritization of variants in rare disease patients remains a challenging task due to the high degree of variability in phenotype presentation and molecular source of disease. Thus, methods that can identify and/or prioritize variants to be clinically reported in the presence of such variability are of critical importance. METHODS: We tested the application of classification algorithms that ingest variant annotations along with phenotype information for predicting whether a variant will ultimately be clinically reported and returned to a patient. To test the classifiers, we performed a retrospective study on variants that were clinically reported to 237 patients in the Undiagnosed Diseases Network. RESULTS: We treated the classifiers as variant prioritization systems and compared them to four variant prioritization algorithms and two single-measure controls. We showed that the trained classifiers outperformed all other tested methods with the best classifiers ranking 72% of all reported variants and 94% of reported pathogenic variants in the top 20. CONCLUSIONS: We demonstrated how freely available binary classification algorithms can be used to prioritize variants even in the presence of real-world variability. Furthermore, these classifiers outperformed all other tested methods, suggesting that they may be well suited for working with real rare disease patient datasets.


Assuntos
Algoritmos , Doenças Genéticas Inatas/diagnóstico , Genômica/métodos , Mutação , Doenças Raras/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Genoma Humano , Humanos , Fenótipo , Polimorfismo Genético , Medicina de Precisão/métodos , Doenças Raras/genética , Estudos Retrospectivos , Análise de Sequência de DNA/métodos , Software
4.
Nat Cell Biol ; 21(9): 1164-1172, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481796

RESUMO

Single-cell measurement of chromatin states, including histone modifications and non-histone protein binding, remains challenging. Here, we present a low-cost, efficient, simultaneous indexing and tagmentation-based ChIP-seq (itChIP-seq) method, compatible with both low cellular input and single cells for profiling chromatin states. itChIP combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation within a single tube, enabling the processing of samples from tens of single cells to, more commonly, thousands of single cells per assay. We demonstrate that single-cell itChIP-seq (sc-itChIP-seq) yields ~9,000 unique reads per cell. Using sc-itChIP-seq to profile H3K27ac, we sufficiently capture the earliest epigenetic priming event during the cell fate transition from naive to primed pluripotency, and reveal the basis for cell-type specific enhancer usage during the differentiation of bipotent cardiac progenitor cells into endothelial cells and cardiomyocytes. Our results demonstrate that itChIP is a widely applicable technology for single-cell chromatin profiling of epigenetically heterogeneous cell populations in many biological processes.


Assuntos
Cromatina/metabolismo , Células Endoteliais/metabolismo , Processamento de Proteína Pós-Traducional/genética , Análise de Sequência de DNA , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina/métodos , Epigenômica/métodos , Histonas/metabolismo , Camundongos Transgênicos , Análise de Sequência de DNA/métodos
5.
Nat Biotechnol ; 37(10): 1229-1236, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501560

RESUMO

The density and long-term stability of DNA make it an appealing storage medium, particularly for long-term data archiving. Existing DNA storage technologies involve the synthesis and sequencing of multiple nominally identical molecules in parallel, resulting in information redundancy. We report the development of encoding and decoding methods that exploit this redundancy using composite DNA letters. A composite DNA letter is a representation of a position in a sequence that consists of a mixture of all four DNA nucleotides in a predetermined ratio. Our methods encode data using fewer synthesis cycles. We encode 6.4 MB into composite DNA, with distinguishable composition medians, using 20% fewer synthesis cycles per unit of data, as compared to previous reports. We also simulate encoding with larger composite alphabets, with distinguishable composition deciles, to show that 75% fewer synthesis cycles are potentially sufficient. We describe applicable error-correcting codes and inference methods, and investigate error patterns in the context of composite DNA letters.


Assuntos
DNA/síntese química , Algoritmos , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Armazenamento e Recuperação da Informação , Análise de Sequência de DNA/métodos
6.
Orv Hetil ; 160(39): 1533-1541, 2019 Sep.
Artigo em Húngaro | MEDLINE | ID: mdl-31544493

RESUMO

Our health is highly determined by the diverse microbial community living within our body and upon our skin. Balance among the members of the commensal microbiota is essential for the preservation of health. New generation sequencing is a rapid, sensitive method for determining the whole microbiome without prior hypothesis and also gives information on the resistance and virulence status. Application of this method can help to identify the pathogens contributing to different diseases, and also the protective bacteria inhibiting their growth. Detecting the changes of the microbiome helps to identify new therapeutic targets and establish targeted antibiotic therapy. Broad-spectrum antibiotics also act against the beneficial members of the microbial flora, which may lead to the development of recurrent or chronic disease. Ear, nose and throat infections are the most common infective diseases in humans and the leading cause for antibiotic prescription worldwide. In recent years, many studies using molecular biology methods were performed examining the microbiome of healthy humans and in otorhinolaryngologic diseases. In the present work, the authors review the changes of the microbiological communities in the healthy state and in various pathologic states in the anatomic regions of the ear, nose and throat. Orv Hetil. 2019; 160(39): 1533-1541.


Assuntos
Bactérias/classificação , Orelha/microbiologia , Microbiota , Nariz/microbiologia , Otolaringologia , Otorrinolaringopatias/microbiologia , Faringe/microbiologia , Antibacterianos/uso terapêutico , Bactérias/genética , Bactérias/patogenicidade , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Humanos , Otolaringologia/tendências , Otorrinolaringopatias/tratamento farmacológico , Otorrinolaringopatias/terapia , Análise de Sequência de DNA/métodos
7.
Arch Virol ; 164(12): 3073-3079, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31555902

RESUMO

A canine parvovirus (CPV)-like virus was detected by PCR and isolated from dead coatis in Argentina. Analysis of the full-length genome sequence revealed that it resembled CPV-but also contained a mutation in the VP2 protein (Arg377Ser) that has not been described previously. This is the first report of a CPV-like virus producing clinical disease in coatis. Genetic similarity to CPV-2c viruses detected in Brazil suggests a strong relationship between these viruses. Although the pathogenic potential of CPV- and feline panleukopenia virus (FPV)-like strains in wild animals is still not completely understood, this study highlights the importance of parvoviruses as a threat to wildlife if proper conditions are present.


Assuntos
Proteínas do Capsídeo/genética , Infecções por Parvoviridae/mortalidade , Parvovirus Canino/classificação , Procyonidae/virologia , Animais , Argentina , Brasil , Tamanho do Genoma , Mutação , Infecções por Parvoviridae/veterinária , Parvovirus Canino/genética , Parvovirus Canino/isolamento & purificação , Filogenia , Análise de Sequência de DNA/métodos
8.
Nat Biotechnol ; 37(10): 1155-1162, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31406327

RESUMO

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the 'genome in a bottle' (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.


Assuntos
DNA Circular/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Variação Genética , Haplótipos , Humanos
9.
Cancer Sci ; 110(10): 3235-3243, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368627

RESUMO

Cytology is widely conducted for diagnosis of urothelial bladder cancer; however, its sensitivity is still low. Recent studies show that liquid biopsies can reflect tumor genomic profiles. We aim to investigate whether plasma or urine is more suitable for detecting tumor-derived DNA in patients with early-stage urothelial bladder cancer. Targeted sequencing of 71 genes was carried out using a total of 150 samples including primary tumor, urine supernatant, urine precipitation, plasma and buffy coat from 25 patients with bladder cancer and five patients with cystitis and benign tumor. We compared mutation profiles between each sample, identified tumor-identical mutations and compared tumor diagnostic sensitivities between urine and conventional cytology. We identified a total of 168 somatic mutations in primary tumor. In liquid biopsies, tumor-identical mutations were found at 53% (89/168) in urine supernatant, 48% (81/168) in urine precipitation and 2% (3/168) in plasma. The high variant allele fraction of urine was significantly related to worse clinical indicators such as tumor invasion and cytological examination. Although conventional cytology detected tumor cells in only 22% of non-invasive tumor, tumor diagnostic sensitivity increased to 67% and 78% using urine supernatant and precipitation, respectively. Urine is an ideal liquid biopsy for detecting tumor-derived DNA and more precisely reflects tumor mutational profiles than plasma. Genomic analysis of urine is clinically useful for diagnosis of superficial bladder cancer at early stage.


Assuntos
Biomarcadores Tumorais/urina , Mutação , Análise de Sequência de DNA/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Urina/química , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Detecção Precoce de Câncer , Feminino , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina
10.
Genome Biol ; 20(1): 153, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375138

RESUMO

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.


Assuntos
Resistência Microbiana a Medicamentos/genética , Metagenômica/métodos , Microbiota/genética , Análise de Sequência de DNA/métodos , Vírus/genética , Animais , Bovinos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transferência Genética Horizontal , Genes Microbianos , Fases de Leitura Aberta , Prófagos/genética , Rúmen/microbiologia , Rúmen/virologia , Vírus/isolamento & purificação
11.
Nat Biotechnol ; 37(8): 907-915, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31375807

RESUMO

The human reference genome represents only a small number of individuals, which limits its usefulness for genotyping. We present a method named HISAT2 (hierarchical indexing for spliced alignment of transcripts 2) that can align both DNA and RNA sequences using a graph Ferragina Manzini index. We use HISAT2 to represent and search an expanded model of the human reference genome in which over 14.5 million genomic variants in combination with haplotypes are incorporated into the data structure used for searching and alignment. We benchmark HISAT2 using simulated and real datasets to demonstrate that our strategy of representing a population of genomes, together with a fast, memory-efficient search algorithm, provides more detailed and accurate variant analyses than other methods. We apply HISAT2 for HLA typing and DNA fingerprinting; both applications form part of the HISAT-genotype software that enables analysis of haplotype-resolved genes or genomic regions. HISAT-genotype outperforms other computational methods and matches or exceeds the performance of laboratory-based assays.


Assuntos
Genótipo , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Software , Sequência de Bases , Benchmarking , Variação Genética , Genoma Humano , Genômica , Humanos , Reprodutibilidade dos Testes
12.
Medicine (Baltimore) ; 98(35): e16626, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464899

RESUMO

Gastric cancer (GC) is one of the common malignant tumors in China, with a high morbidity and mortality. With the development and application of high-throughput sequencing technologies and metagenomics, a great quantity of studies have shown that gastrointestinal microbiota is closely related to digestive system diseases. Although some studies have reported the effect of long-term follow-up after subtotal gastrectomy on intestinal flora changes in patients with GC. However, the features of gut microbiota and their shifts in patients with GC in perioperative period remain unclear.This study was designed to characterize fecal microbiota shifts of the patients with GC before and after the radical distal gastrectomy (RDG) during their hospital staying periods. Furthermore, fecal microbiota was also compared between the GC patients and healthy individuals.Patients who were diagnosed with advanced gastric adenocarcinoma at distal stomach were enrolled in the study. The bacterial burden within fecal samples was determined using quantitative polymerase chain reaction. To analyze the diversity and composition of gut microbiota from fecal DNA of 20 GC patients and 22 healthy controls, amplicons of the 16S rRNA gene from all subjects were pyrosequenced. To study gut microbiota shifts, the fecal microbiota from 6 GC patients before and after RDG was detected and subsequently analyzed. Short-chain fatty acids were also detected by chromatography spectrometer in these 6 GC patients.RDG had a moderate effect on bacterial richness and evenness, but had pronounced effects on the composition of postoperative gut microbiota compared with preoperative group. The relative abundances of genera Akkermansia, Esherichia/Shigella, Lactobacillus, and Dialister were significant changed in perioperative period. Remarkably, higher abundances of Escherichia/Shigella, Veillonella, and Clostridium XVIII and lower abundances of Bacteroides were observed in gut microbiota of overall GC patients compared to healthy controls.This study is the first study to characterize the altered gut microbiota within fecal samples from GC patients during perioperative period, and provide a new insights on such microbial perturbations as a potential effector of perioperative period phenotype. Further research must validate these discoveries and may evaluate targeted microbiota shifts to improve outcomes in GC patients.


Assuntos
Bactérias/classificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Neoplasias Gástricas/cirurgia , Adulto , Bactérias/genética , Bactérias/isolamento & purificação , China , DNA Bacteriano/genética , DNA Ribossômico/genética , Feminino , Gastrectomia , Microbioma Gastrointestinal , Humanos , Masculino , Pessoa de Meia-Idade , Período Perioperatório , Filogenia , Neoplasias Gástricas/microbiologia
13.
BMC Bioinformatics ; 20(1): 431, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426747

RESUMO

BACKGROUND: Protein pulldown using Methyl-CpG binding domain (MBD) proteins followed by high-throughput sequencing is a common method to determine DNA methylation. Algorithms have been developed to estimate absolute methylation level from read coverage generated by affinity enrichment-based techniques, but the most accurate one for MBD-seq data requires additional data from an SssI-treated Control experiment. RESULTS: Using our previous characterizations of Methyl-CpG/MBD2 binding in the context of an MBD pulldown experiment, we build a model of expected MBD pulldown reads as drawn from SssI-treated DNA. We use the program BayMeth to evaluate the effectiveness of this model by substituting calculated SssI Control data for the observed SssI Control data. By comparing methylation predictions against those from an RRBS data set, we find that BayMeth run with our modeled SssI Control data performs better than BayMeth run with observed SssI Control data, on both 100 bp and 10 bp windows. Adapting the model to an external data set solely by changing the average fragment length, our calculated data still informs the BayMeth program to a similar level as observed data in predicting methylation state on a pulldown data set with matching WGBS estimates. CONCLUSION: In both internal and external MBD pulldown data sets tested in this study, BayMeth used with our modeled pulldown coverage performs better than BayMeth run without the inclusion of any estimate of SssI Control pulldown, and is comparable to - and in some cases better than - using observed SssI Control data with the BayMeth program. Thus, our MBD pulldown alignment model can improve methylation predictions without the need to perform additional control experiments.


Assuntos
Biologia Computacional/métodos , Metilação de DNA/genética , DNA-Citosina Metilases/metabolismo , DNA/metabolismo , Modelos Biológicos , Alinhamento de Sequência , Algoritmos , Pareamento de Bases , Cromossomos Humanos Par 7/genética , Ilhas de CpG/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Domínio de Ligação a CpG Metilada , Análise de Sequência de DNA/métodos
14.
Cancer Sci ; 110(10): 3375-3381, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31436356

RESUMO

Cell-free DNA (cfDNA) analysis to detect circulating tumor DNA has been focused on monitoring malignant lymphomas. However, clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations can also be detected by cfDNA analysis. Our aim is to investigate the origin of mutations detected in cfDNA among B-cell lymphoma patients. MYD88/CD79B, DNMT3A, and TP53 were chosen as genes of interest, representing each of the following categories: lymphoma driver genes, CHIP-related genes, and genes shared between lymphoma and CHIP. Seventy-five B-cell lymphoma patients were included in this retrospective study. Serum cfDNAs at time of complete metabolic response (CMR) were sequenced for TP53 (N = 75) and DNMT3A (N = 49). MYD88 p.L265P and CD79B p.Y196C/H mutations were analyzed in diffuse large B-cell lymphoma (DLBCL) patients whose tumor samples were available (N = 29). Two and seven mutations in TP53 and DNMT3A, respectively, were detected in cfDNA at CMR. These mutations were detected in either bone marrow mononuclear cells (BMMC) or PBMC. Although four DNMT3A mutations were also detected in tumors, median variant allele frequencies in the tumors (<1.0%) were significantly lower than those in both BMMC (6.1%) and serum (5.2%) obtained before the therapy. Conversely, five MYD88 and three CD79B mutations detected in tumors were confirmed in cfDNA before therapy, but not in BMMC nor in cfDNA at CMR. Thus, all TP53 and DNMT3A mutations detected in cfDNA at remission seemed to originate from CHIP rather than from residual disease. Results of liquid biopsy should be carefully interpreted, especially in genes shared between lymphomas and CHIP.


Assuntos
Células Clonais/química , DNA (Citosina-5-)-Metiltransferases/genética , Hematopoese , Linfoma de Células B/genética , Mutação , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/química , Ácidos Nucleicos Livres/genética , Feminino , Frequência do Gene , Humanos , Leucócitos Mononucleares/química , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Monócitos/química , Indução de Remissão , Estudos Retrospectivos , Análise de Sequência de DNA/métodos
15.
Cancer Sci ; 110(10): 3368-3374, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432574

RESUMO

BRCA1/2 genes are the most frequently germline mutated DNA-repair genes, and the survival of BRCA1/2 carriers has been extensively explored in breast cancer. However, the prevalence of germline mutations in non-BRCA1/2 DNA-repair genes and the survival of carriers are largely unknown in a large cohort of unselected breast cancer patients. Germline mutations in 16 DNA-repair genes were determined using a multigene panel in 7657 BRCA1/2-negative breast cancer patients who were unselected for family history of cancer or age at diagnosis. Among the 7657 BRCA1/2-negative breast cancer patients, 257 (3.4%) carried at least 1 pathogenic germline mutation in the 16 DNA-repair genes. The prevalence of DNA-repair gene mutations was significantly higher in familial breast cancers (5.2%, P = 0.002) and early-onset breast cancers (diagnosed at and before the age of 40) (4.5%, P = 0.003) than that of sporadic breast cancers (2.9%) (diagnosed above age of 40), respectively. The DNA-repair gene mutation carriers were significantly more likely to have a larger tumor (P = 0.04) and axillary lymph node metastasis (P = 0.03). Moreover, DNA-repair gene mutation was an independent unfavorable factor for recurrence-free survival (adjusted hazard ratio [HR] = 1.38, 95% CI: 1.00-1.91, P = 0.05) and disease-specific survival (adjusted HR=1.63, 95% CI: 1.04-2.57, P = 0.03) in this cohort. Overall, 3.4% of BRCA1/2-negative breast cancer patients carried germline mutations in the 16 DNA-repair genes, and the DNA-repair gene mutation carriers exhibited an aggressive phenotype and had poor survival compared with noncarriers.


Assuntos
Neoplasias da Mama/patologia , Reparo do DNA , Mutação em Linhagem Germinativa , Metástase Linfática/patologia , Análise de Sequência de DNA/métodos , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Feminino , Predisposição Genética para Doença , Humanos , Metástase Linfática/genética , Pessoa de Meia-Idade , Análise de Sobrevida , Carga Tumoral , Adulto Jovem
16.
Lancet ; 394(10197): 533-540, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31395441

RESUMO

One of the primary goals of genomic medicine is to improve diagnosis through identification of genomic conditions, which could improve clinical management, prevent complications, and promote health. We explore how genomic medicine is being used to obtain molecular diagnoses for patients with previously undiagnosed diseases in prenatal, paediatric, and adult clinical settings. We focus on the role of clinical genomic sequencing (exome and genome) in aiding patients with conditions that are undiagnosed even after extensive clinical evaluation and testing. In particular, we explore the impact of combining genomic and phenotypic data and integrating multiple data types to improve diagnoses for patients with undiagnosed diseases, and we discuss how these genomic sequencing diagnoses could change clinical management.


Assuntos
Doenças Raras/diagnóstico , Análise de Sequência de DNA/métodos , Adulto , Criança , Diagnóstico Precoce , Genômica , Humanos , Fenótipo , Diagnóstico Pré-Natal/métodos , Doenças Raras/genética , Sequenciamento Completo do Exoma , Sequenciamento Completo do Genoma
18.
Genome Biol ; 20(1): 134, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287019

RESUMO

We present SMURF-seq, a protocol to efficiently sequence short DNA molecules on a long-read sequencer by randomly ligating them to form long molecules. Applying SMURF-seq using the Oxford Nanopore MinION yields up to 30 fragments per read, providing an average of 6.2 and up to 7.5 million mappable fragments per run, increasing information throughput for read-counting applications. We apply SMURF-seq on the MinION to generate copy number profiles. A comparison with profiles from Illumina sequencing reveals that SMURF-seq attains similar accuracy. More broadly, SMURF-seq expands the utility of long-read sequencers for read-counting applications.


Assuntos
Variações do Número de Cópias de DNA , Análise de Sequência de DNA/métodos , Linhagem Celular Tumoral , Feminino , Humanos
19.
Nat Commun ; 10(1): 3066, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296857

RESUMO

Metagenomic sequence classification should be fast, accurate and information-rich. Emerging long-read sequencing technologies promise to improve the balance between these factors but most existing methods were designed for short reads. MetaMaps is a new method, specifically developed for long reads, capable of mapping a long-read metagenome to a comprehensive RefSeq database with >12,000 genomes in <16 GB or RAM on a laptop computer. Integrating approximate mapping with probabilistic scoring and EM-based estimation of sample composition, MetaMaps achieves >94% accuracy for species-level read assignment and r2 > 0.97 for the estimation of sample composition on both simulated and real data when the sample genomes or close relatives are present in the classification database. To address novel species and genera, which are comparatively harder to predict, MetaMaps outputs mapping locations and qualities for all classified reads, enabling functional studies (e.g. gene presence/absence) and detection of incongruities between sample and reference genomes.


Assuntos
Biologia Computacional/métodos , Análise de Dados , Metagenômica/métodos , Algoritmos , Conjuntos de Dados como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenoma/genética , Microbiota/genética , Análise de Sequência de DNA/métodos , Software
20.
Gene ; 713: 143971, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31299361

RESUMO

An in silico genome analysis of the probiotic Bacillus strain FTC01 was performed. The draft genome comprises 3.9 Mb, with a G + C content of 46.6% and a total of 3941 coding sequences. The species of strain FTC01 was defined as B. velezensis during GenBank genome annotation, following the current nomenclature. Eight gene clusters involved in the synthesis of non-ribosomal lipopeptides, polyketides and bacilysin were found, as well as part of the gene cluster involved in the synthesis of cyclic lipopeptide locillomycin. The production of lipopeptides surfactin and iturin by strain FTC01 was confirmed. In addition, a gene encoding a peptidylprolyl isomerase, involved in bacterial adhesion to the host tissue, beyond twelve genes responsible for acid tolerance and several hydrolase genes were found. These characteristics may help in host colonization and maintenance and may account for the probiotic properties observed for strain FTC01.


Assuntos
Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Genoma Bacteriano , Metaboloma , Probióticos/metabolismo , Bacillus/crescimento & desenvolvimento , DNA Bacteriano/análise , Filogenia , Análise de Sequência de DNA/métodos
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