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2.
Breast ; 45: 29-35, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30822622

RESUMO

Multigene panel testing for breast and ovarian cancer predisposition diagnosis is a useful tool as it makes possible to sequence a considerable number of genes in a large number of individuals. More than 200 different multigene panels in which the two major BRCA1 and BRCA2 breast cancer predisposing genes are included are proposed by public or commercial laboratories. We review the clinical validity and clinical utility of the 26 genes most oftenly included in these panels. Because clinical validity and utility are not established for all genes and due to the heterogeneity of tumour risk levels, there is a substantial difficulty in the routine use of multigene panels if management guidelines and recommendations for testing relatives are not previously defined for each gene. Besides, the classification of variant of unknown significance (VUS) is a particular limitation and challenge. Efforts to classify VUSs and also to identify factors that modify cancer risks are now needed to produce personalised risk estimates. The complexity of information, the capacity to come back to patients when VUS are re-classified as pathogenic, and the expected large increase in the number of individuals to be tested especially when the aim of multigene panel testing is not only prevention but also treatment are challenging both for physicians and patients. Quality of tests, interpretation of results, information and accompaniment of patients must be at the heart of the guidelines of multigene panel testing.


Assuntos
Neoplasias da Mama/genética , Detecção Precoce de Câncer/normas , Predisposição Genética para Doença , Testes Genéticos/normas , Análise de Sequência/normas , Biomarcadores Tumorais/genética , Detecção Precoce de Câncer/métodos , Feminino , Genes BRCA1 , Genes BRCA2 , Testes Genéticos/métodos , Variação Genética , Humanos , Neoplasias Ovarianas/genética , Reprodutibilidade dos Testes , Análise de Sequência/métodos
3.
J Dairy Sci ; 102(5): 3912-3923, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30852020

RESUMO

Traditional fermented dairy foods have been the major components of the Mongolian diet for millennia. In this study, we used propidium monoazide (PMA; binds to DNA of nonviable cells so that only viable cells are enumerated) and single-molecule real-time sequencing (SMRT) technology to investigate the total and viable bacterial compositions of 19 traditional fermented dairy foods, including koumiss from Inner Mongolia (KIM), koumiss from Mongolia (KM), and fermented cow milk from Mongolia (CM); sample groups treated with PMA were designated PKIM, PKM, and PCM. Full-length 16S rRNA sequencing identified 195 bacterial species in 121 genera and 13 phyla in PMA-treated and untreated samples. The PMA-treated and untreated samples differed significantly in their bacterial community composition and α-diversity values. The predominant species in KM, KIM, and CM were Lactobacillus helveticus, Streptococcus parauberis, and Lactobacillus delbrueckii, whereas the predominant species in PKM, PKIM, and PCM were Enterobacter xiangfangensis, Lactobacillus helveticus, and E. xiangfangensis, respectively. Weighted and unweighted principal coordinate analyses showed a clear clustering pattern with good separation and only minor overlapping. In addition, a pure culture method was performed to obtain lactic acid bacteria resources in dairy samples according to the results of SMRT sequencing. A total of 102 LAB strains were identified and Lb. helveticus (68.63%) was the most abundant, in agreement with SMRT sequencing results. Our results revealed that the bacterial communities of traditional dairy foods are complex and vary by type of fermented dairy product. The PMA treatment induced significant changes in bacterial community structure.


Assuntos
Azidas , Produtos Fermentados do Leite/microbiologia , Microbiota , Propídio/análogos & derivados , Análise de Sequência/métodos , Animais , Bactérias/classificação , Bovinos , China , DNA Bacteriano/análise , Feminino , Fermentação , Kumis , Lactobacillales/genética , Lactobacillus delbrueckii/genética , Lactobacillus helveticus/genética , Leite/microbiologia , Mongólia , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética
4.
Mol Oral Microbiol ; 34(2): 39-50, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30739386

RESUMO

Querying gene function in bacteria has been greatly accelerated by the advent of transposon sequencing (Tn-seq) technologies (related Tn-seq strategies are known as TraDIS, INSeq, RB-TnSeq, and HITS). Pooled populations of transposon mutants are cultured in an environment and next-generation sequencing tools are used to determine areas of the genome that are important for bacterial fitness. In this review we provide an overview of Tn-seq methodologies and discuss how Tn-seq has been applied, or could be applied, to the study of oral microbiology. These applications include studying the essential genome as a means to rationally design therapeutic agents. Tn-seq has also contributed to our understanding of well-studied biological processes in oral bacteria. Other important applications include in vivo pathogenesis studies and use of Tn-seq to probe the molecular basis of microbial interactions. We also highlight recent advancements in techniques that act in synergy with Tn-seq such as clustered regularly interspaced short palindromic repeats (CRISPR) interference and microfluidic chip platforms.


Assuntos
Bactérias/genética , Elementos de DNA Transponíveis/genética , Genes Essenciais/genética , Boca/microbiologia , Análise de Sequência de DNA/métodos , Análise de Sequência/métodos , Sistemas de Liberação de Medicamentos , Genes Essenciais/efeitos dos fármacos , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Interações Microbianas/genética , Mutagênese Insercional , Fenótipo , Análise de Sequência/instrumentação , Análise de Sequência de DNA/instrumentação
5.
Dis Colon Rectum ; 62(4): 429-437, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30730459

RESUMO

BACKGROUND: Genomic profiling of colorectal cancer aims to identify actionable somatic mutations but can also discover incidental germline findings. OBJECTIVE: The purpose of this study was to report the detection of pathogenic germline variants that confer heritable cancer predisposition. DESIGN: This was a retrospective study. SETTINGS: The study was conducted at a tertiary-referral institution. PATIENTS: Between 2012 and 2015, 1000 patients with advanced cancer underwent targeted exome sequencing of a 202-gene panel. The subgroup of 151 patients with advanced colorectal cancer who underwent matched tumor-normal (blood) sequencing formed our study cohort. INTERVENTIONS: Germline variants in 46 genes associated with hereditary cancer predisposition were classified according to a defined algorithm based on in silico predictions of pathogenicity. Patients with presumed pathogenic variants were examined for type of mutation, as well as clinical, pedigree, and clinical genetic testing data. MAIN OUTCOME MEASURES: We measured detection of pathogenic germline variants. RESULTS: A total of 1910 distinct germline variants were observed in 151 patients. After filtering, 15 pathogenic germline variants (9.9%) were found in 15 patients, arising from 9 genes of varying penetrance for colorectal cancer (APC (n = 2; 13%), ATM (n = 1; 6%), BRCA1 (n = 2; 13%), CDH1 (n = 2; 13%), CHEK2 (n = 4; 27%), MSH2 (n = 1; 7%), MSH6 (n = 1; 7%), NF2 (n = 1; 7%), and TP53 (n = 1; 7%)). Patients with pathogenic variants were diagnosed at a younger age than those without (median, 45 vs 52 y; p = 0.03). Of the 15 patients, 7 patients (46.7%) with variants in low/moderate- penetrant genes for colorectal cancer would likely have not been tested based on clinical and pedigree criteria, where 2 harbored clinically actionable variants (CDH1 and NF2, 28.5% of 7). LIMITATIONS: This study was limited by its small sample size and advanced-stage patients. CONCLUSIONS: Tumor-normal sequencing can incidentally discover clinically unsuspected germline variants that confer cancer predisposition in 9.9% of patients with advanced colorectal cancer. Precision medicine should integrate clinical cancer genetics to inform and interpret the actionability of germline variants and to provide follow-up care to mutation carriers. See Video Abstract at http://links.lww.com/DCR/A906.


Assuntos
Neoplasias Colorretais , Adulto , Fatores Etários , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Testes Genéticos/métodos , Genômica/métodos , Mutação em Linhagem Germinativa , Humanos , Achados Incidentais , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Medicina de Precisão/métodos , Estudos Retrospectivos , Análise de Sequência/métodos , Estados Unidos
6.
PLoS One ; 14(2): e0209523, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30759172

RESUMO

We present elPrep 4, a reimplementation from scratch of the elPrep framework for processing sequence alignment map files in the Go programming language. elPrep 4 includes multiple new features allowing us to process all of the preparation steps defined by the GATK Best Practice pipelines for variant calling. This includes new and improved functionality for sorting, (optical) duplicate marking, base quality score recalibration, BED and VCF parsing, and various filtering options. The implementations of these options in elPrep 4 faithfully reproduce the outcomes of their counterparts in GATK 4, SAMtools, and Picard, even though the underlying algorithms are redesigned to take advantage of elPrep's parallel execution framework to vastly improve the runtime and resource use compared to these tools. Our benchmarks show that elPrep executes the preparation steps of the GATK Best Practices up to 13x faster on WES data, and up to 7.4x faster for WGS data compared to running the same pipeline with GATK 4, while utilizing fewer compute resources.


Assuntos
Análise de Sequência/métodos , Algoritmos , Biologia Computacional/economia , Biologia Computacional/métodos , Custos e Análise de Custo , Exoma , Análise de Sequência/economia , Software
7.
Medicine (Baltimore) ; 98(4): e14157, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30681580

RESUMO

RATIONALE: Hypochondroplasia (HCH) is the mildest form of chondrodysplasia characterized by disproportionate short stature, short extremities, and variable lumbar lordosis. It is caused by mutations in fibroblast growth factor receptor 3 (FGFR3) gene. Up to date, at least thirty mutations of FGFR3 gene have been found to be related to HCH. However, mutational screening of the FGFR3 gene is still far from completeness. Identification of more mutations is particularly important in diagnosis of HCH and will gain more insights into the molecular basis for the pathogenesis of HCH. PATIENT CONCERNS: A large Chinese family consisting of 53 affected individuals with HCH phenotypes was examined. DIAGNOSES: A novel missense mutation, c.1052C>T, in FGFR3 gene was identified in a large Chinese family with HCH. On the basis of this finding and clinical manifestations, the final diagnosis of HCH was made. INTERVENTIONS: Next-generation sequencing (NGS) of DNA samples was performed to detect the mutation in the chondrodysplasia-related genes on the proband and her parents, which was confirmed by Sanger sequencing in the proband and most of other living affected family members. OUTCOMES: A novel missense mutation, c.1052C>T, in the extracellular, ligand-binding domain of FGFR3 was identified in a large Chinese family with HCH. This heterozygous mutation results in substitution of serine for phenylalanine at amino acid 351 (p.S351F) and co-segregates with the phenotype in this family. Molecular docking analysis reveals that this unique FGFR3 mutation results in an enhancement of ligand-binding affinity between FGFR3 and its main ligand, fibroblast growth factor 9. LESSONS: This novel mutation is the first mutation displaying an increase in ligand-binding affinity, therefore it may serve as a model to investigate ligand-dependent activity of FGF-FGFR complex. Our data also expanded the mutation spectrum of FGFR3 gene and facilitated clinic diagnosis and genetic counseling for this family with HCH.


Assuntos
Osso e Ossos/anormalidades , Nanismo/genética , Deformidades Congênitas dos Membros/genética , Lordose/genética , Mutação de Sentido Incorreto/genética , Linhagem , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Análise de Sequência/métodos , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Criança , Feminino , Frequência do Gene , Heterozigoto , Humanos , Masculino , Simulação de Acoplamento Molecular , Fenótipo
8.
Medicine (Baltimore) ; 97(50): e13565, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30558019

RESUMO

Dravet syndrome is considered to be one of the most severe types of genetic epilepsy. Mutations in SCN1A gene have been found to be responsible for at least 80% of patients with Dravet syndrome, and 90% of these mutations arise de novo. The variable clinical phenotype is commonly observed among these patients with SCN1A mutations, suggesting that genetic modifiers may influence the phenotypic expression of Dravet syndrome. In the present study, we described the clinical, pathological, and molecular characteristics of 13 Han Chinese pedigrees clinically diagnosed with Dravet syndrome. By targeted-exome sequencing, bioinformatics analysis and Sanger sequencing verification, 11 variants were identified in SCN1A gene among 11 pedigrees including 7 missense mutations, 2 splice site mutations, and 2 frameshift mutations (9 novel variants and 2 reported mutations). Particularly, 2 of these Dravet syndrome patients with SCN1A variants also harbored SCN9A, KCNQ2, or SLC6A8 variants. In addition, 2 subjects were failed to detect any pathogenic mutations in SCN1A and other epilepsy-related genes. These data suggested that SCN1A variants account for about 84.6% of Dravet syndrome in our cohort. This study expanded the mutational spectrum for the SCN1A gene, and also provided clinical and genetic evidence for the hypothesis that genetic modifiers may contribute to the variable manifestation of Dravet syndrome patients with SCN1A mutations. Thus, targeted-exome sequencing will make it possible to detect the interactions of epilepsy-related genes and reveal their modification on the severity of SCN1A mutation-related Dravet syndrome.


Assuntos
Epilepsias Mioclônicas/genética , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Linhagem , Análise de Sequência/métodos , Grupo com Ancestrais do Continente Asiático/genética , Pré-Escolar , China , Feminino , Mutação da Fase de Leitura/genética , Humanos , Lactente , Canal de Potássio KCNQ2/genética , Masculino , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Proteínas do Tecido Nervoso/genética , Fenótipo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Isoformas de Proteínas/genética
9.
Curr Microbiol ; 75(12): 1649-1654, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30267141

RESUMO

Two strains of Pseudomonas putida, Psp-LUP and Psp-SPAR, capable of growth on the quinolizidine alkaloids, lupanine and sparteine respectively, were studied here. We report the isolation of Psp-SPAR and the complete genome sequencing of both bacteria. Both were confirmed to belong to P. putida, Psp-LUP close to the type isolate of the species (NBRC14164T) and Psp-SPAR close to strains KT2440 and F1. Psp-SPAR did not grow on lupanine but did contain a gene encoding a putative quinolizidine-17-hydroxylase peptide which exhibited high similarity (76%identity) to the lupanine-17-hydroxylase characterised from Psp-LUP.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Pseudomonas putida/genética , Esparteína/genética , Esteroide 17-alfa-Hidroxilase/genética , Alcaloides/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Análise de Sequência/métodos , Esparteína/análogos & derivados , Sequenciamento Completo do Genoma/métodos
10.
Med. clín (Ed. impr.) ; 151(2): 80.e1-80.e10, jul. 2018. tab
Artigo em Espanhol | IBECS | ID: ibc-173778

RESUMO

El diagnóstico genético de los síndromes de cáncer hereditario ofrece la oportunidad de establecer unas medidas de predicción/prevención eficaces en el paciente y sus familiares que se traducen en una disminución de la morbimortalidad por cáncer en las familias de alto riesgo genético. La secuenciación masiva (NGS) ofrece una considerable mejora de la eficiencia del diagnóstico genético, permitiendo un aumento del rendimiento diagnóstico con una reducción sustancial del tiempo de respuesta y costes económicos. En consecuencia, la implementación de esta nueva tecnología es una gran oportunidad de mejora en el manejo clínico de las familias afectas. El objetivo de la presente guía es establecer un marco de recomendaciones útiles para una implementación planificada y controlada de la NGS en el contexto de la predisposición hereditaria a cáncer, que permita potenciar las fortalezas y oportunidades que ofrece dicha tecnología y minimizar las debilidades y amenazas que puedan derivarse de su uso. Está inspirada en las recomendaciones de sociedades internacionales, habiendo sido adaptada a nuestro entorno, y teniendo en cuenta aspectos coyunturales a nivel organizativo y biojurídico. Se aportan 41 declaraciones agrupadas en 6 apartados: utilidad clínica y diagnóstica, consentimiento informado y asesoramiento genético pretest y postest, validación de los procedimientos analíticos, informe de resultados, gestión de la información y distinción entre ámbito de investigación y ámbito asistencial. Esta guía ha sido elaborada por la Asociación Española de Genética Humana (AEGH), la Sociedad Española de Medicina de Laboratorio (SEQC-ML) y la Sociedad Española de Oncología Médica (SEOM)


Genetic diagnosis of hereditary cancer syndromes offers the opportunity to establish more effective predictive and preventive measures for the patient and their families. The ultimate objective is to decrease cancer morbidity and mortality in high genetic risk families. Next Generation Sequencing (NGS) offers an important improvement in the efficiency of genetic diagnosis, allowing an increase in diagnostic yield with a substantial reduction in response times and economic costs. Consequently, the implementation of this new technology is a great opportunity for improvement in the clinical management of affected families. The aim of these guidelines is to establish a framework of useful recommendations for planned and controlled implementation of NGS in the context of hereditary cancer. These will help to consolidate the strengths and opportunities offered by this technology, and minimise the weaknesses and threats which may derive from its use. The recommendations of international societies have been adapted to our environment, taking the Spanish context into account at organisational and juridical levels. Forty-one statements are grouped under six headings: clinical and diagnostic utility, informed consent and genetic counselling pre-test and post-test, validation of analytical procedures, results report, management of information and distinction between research and clinical context. This guide has been developed by the Spanish Association of Human Genetics (AEGH), the Spanish Society of Laboratory Medicine (SEQC-ML) and the Spanish Society of Medical Oncology (SEOM)


Assuntos
Neoplasias/genética , Análise de Sequência/métodos , Neoplasias/diagnóstico , Testes Genéticos/métodos , Guias de Prática Clínica como Assunto
11.
BMC Res Notes ; 11(1): 360, 2018 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880035

RESUMO

OBJECTIVES: The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shiga toxin (Stx) and the E. coli outer membrane protein (Eae) or intimin, encoded by the stx and eae genes, respectively. Although several polymerase chain reaction (PCR) protocols target these virulence genes, few aim at detecting all variants or have an internal amplification control (IAC) included in a multiplex assay. The objective of this work was to develop a simple multiplex PCR assay in order to detect all stx and eae variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. RESULTS: The wecA gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of stx and eae were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the stx genes was achieved by subjecting the PCR products to restriction digestion and semi-nested duplex PCR, providing a simple screening assay for human diarrhoea diagnostic.


Assuntos
Adesinas Bacterianas/genética , Antígenos de Bactérias/genética , Diarreia/diagnóstico , Enterobacteriaceae/genética , Proteínas de Escherichia coli/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência/métodos , Toxina Shiga I/genética , Toxina Shiga II/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Humanos
12.
BMC Genomics ; 19(1): 332, 2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739332

RESUMO

BACKGROUND: Here we present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq). We also present a remediation method that minimizes the impact of such swaps. RESULTS: Leveraging data collected over a two-year period, we demonstrate the widespread prevalence of index swapping in patterned flow cell data. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different library methods can have vastly different swapping rates and that even non-ExAmp chemistry instruments display trace levels of index swapping. We provide methods for eliminating sample data cross contamination by utilizing non-redundant dual indexing for complete filtering of index swapped reads, and share the sequences for 96 non-combinatorial dual indexes we have validated across various library preparation methods and sequencer models. Finally, using computational methods we provide a greater insight into the mechanism of index swapping. CONCLUSIONS: Index swapping in pooled libraries is a prevalent phenomenon that we observe at a rate of 0.2 to 6% in all sequencing runs on HiSeqX, HiSeq 4000/3000, and NovaSeq. Utilizing non-redundant dual indexing allows for the removal (flagging/filtering) of these swapped reads and eliminates swapping induced sample contamination, which is critical for sensitive applications such as RNA-seq, single cell, blood biopsy using circulating tumor DNA, or clinical sequencing.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência/métodos , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Biblioteca Gênica , Genoma Humano , Humanos , Análise de Sequência de DNA
13.
Crit Rev Biotechnol ; 38(8): 1157-1175, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29631431

RESUMO

For more than a quarter of a century, sequencing technologies from Sanger's method to next-generation high-throughput techniques have provided fascinating opportunities in the life sciences. The continuing upward trajectory of sequencing technologies will improve livestock research and expedite the development of various new genomic and technological studies with farm animals. The use of high-throughput technologies in livestock research has increased interest in metagenomics, epigenetics, genome-wide association studies, and identification of single nucleotide polymorphisms and copy number variations. Such studies are beginning to provide revolutionary insights into biological and evolutionary processes. Farm animals, such as cattle, swine, and horses, have played a dual role as economically and agriculturally important animals as well as biomedical research models. The first part of this study explores the current state of sequencing methods, many of which are already used in animal genomic studies, and the second part summarizes the state of cattle, swine, horse, and chicken genome sequencing and illustrates its achievements during the last few years. Finally, we describe several high-throughput sequencing approaches for the improved detection of known, unknown, and emerging infectious agents, leading to better diagnosis of infectious diseases. The insights from viral metagenomics and the advancement of next-generation sequencing will strongly support specific and efficient vaccine development and provide strategies for controlling infectious disease transmission among animal populations and/or between animals and humans. However, prospective sequencing technologies will require further research and in-field testing before reaching the marketplace.


Assuntos
Doenças dos Animais/genética , Análise de Sequência/métodos , Animais , Genômica
14.
Biochem Soc Trans ; 46(3): 491-501, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29626147

RESUMO

Nucleosomes are the unitary structures of chromosome folding, and their arrangements are intimately coupled to the regulation of genome activities. Conventionally, structural analyses using electron microscopy and X-ray crystallography have been used to study such spatial nucleosome arrangements. In contrast, recent improvements in the resolution of sequencing-based methods allowed investigation of nucleosome arrangements separately at each genomic locus, enabling exploration of gene-dependent regulation mechanisms. Here, we review recent studies on nucleosome folding in chromosomes from these two methodological perspectives: conventional structural analyses and DNA sequencing, and discuss their implications for future research.


Assuntos
Genoma , Nucleossomos/metabolismo , Cristalografia por Raios X , Microscopia Eletrônica/métodos , Nucleossomos/química , Análise de Sequência/métodos
15.
Mol Ecol Resour ; 18(4): 825-837, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29633534

RESUMO

High-density genome-wide sequencing increases the likelihood of discovering genes of major effect and genomic structural variation in organisms. While there is an increasing availability of reference genomes across broad taxa, the greatest limitation to whole-genome sequencing of multiple individuals continues to be the costs associated with sequencing. To alleviate excessive costs, pooling multiple individuals with similar phenotypes and sequencing the homogenized DNA (Pool-Seq) can achieve high genome coverage, but at the loss of individual genotypes. Although Pool-Seq has been an effective method for association mapping in model organisms, it has not been frequently utilized in natural populations. To extend bioinformatic tools for rapid implementation of Pool-Seq data in nonmodel organisms, we developed a pipeline called PoolParty and illustrate its effectiveness in genetic association mapping. Alignment expectations based on five pooled Chinook salmon (Oncorhynchus tshawytscha) libraries showed that approximately 48% genome coverage per library could be achieved with reasonable sequencing effort. We additionally examined male and female O. tshawytscha libraries to illustrate how Pool-Seq techniques can successfully map known genes associated with functional differences among sexes such as growth hormone 2. Finally, we compared pools of individuals of different spawning ages for each sex to discover novel genes involved with age at maturity in O. tshawytscha such as opsin4 and transmembrane protein19. While not appropriate for every system, Pool-Seq data processed by the PoolParty pipeline is a practical method for identifying genes of major effect in nonmodel organisms when high genome coverage is necessary and cost is a limiting factor.


Assuntos
Estudos de Associação Genética/métodos , Salmão/genética , Animais , Mapeamento Cromossômico/métodos , Biologia Computacional , Feminino , Genoma , Masculino , Alinhamento de Sequência , Análise de Sequência/métodos , Caracteres Sexuais , Software
16.
PLoS One ; 13(4): e0195601, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29684052

RESUMO

A significant approach for the discovery of biological regulatory rules of genes, protein and their inheritance relationships is the extraction of meaningful patterns from biological sequence data. The existing algorithms of sequence pattern discovery, like MSPM and FBSB, suffice their low efficiency and accuracy. In order to deal with this issue, this paper presents a new algorithm for biological sequence pattern mining abbreviated MpBsmi based on the data index structure. The MpBsmi algorithm employs a sequence position table abbreviated ST and a sequence database index structure named DB-Index for data storing, mining and pattern expansion. The ST and DB-Index of single items are firstly obtained through scanning sequence database once. Then a new algorithm for fast support counting is developed to mine the table ST to identify the frequent single items. Based on a connection strategy, the frequent patterns are expanded and the expanded table ST is updated by scanning the DB-Index. The fast support counting algorithm is used for obtaining the frequent expansion patterns. Finally, a new pruning technique is developed for extended pattern to avoid the generation of unnecessarily large number of candidate patterns. The experiments results on multiple classical protein sequences from the Pfam database validate the performance of the proposed algorithm including the accuracy, stability and scalability. It is showed that the proposed algorithm has achieved the better space efficiency, stability and scalability comparing with MSPM, FBSB which are the two main algorithms for biological sequence mining.


Assuntos
Algoritmos , Mineração de Dados/métodos , Reconhecimento Automatizado de Padrão/métodos , Análise de Sequência/métodos , Animais , DNA/genética , Proteínas/genética , RNA/genética , Fatores de Tempo
17.
Nature ; 556(7699): 108-112, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29590089

RESUMO

Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and cell identity at single-cell resolution, which has been a major challenge. Clonal history has traditionally been investigated by microscopically tracking cells during development, monitoring the heritable expression of genetically encoded fluorescent proteins and, more recently, using next-generation sequencing technologies that exploit somatic mutations, microsatellite instability, transposon tagging, viral barcoding, CRISPR-Cas9 genome editing and Cre-loxP recombination. Single-cell transcriptomics provides a powerful platform for unbiased cell-type classification. Here we present ScarTrace, a single-cell sequencing strategy that enables the simultaneous quantification of clonal history and cell type for thousands of cells obtained from different organs of the adult zebrafish. Using ScarTrace, we show that a small set of multipotent embryonic progenitors generate all haematopoietic cells in the kidney marrow, and that many progenitors produce specific cell types in the eyes and brain. In addition, we study when embryonic progenitors commit to the left or right eye. ScarTrace reveals that epidermal and mesenchymal cells in the caudal fin arise from the same progenitors, and that osteoblast-restricted precursors can produce mesenchymal cells during regeneration. Furthermore, we identify resident immune cells in the fin with a distinct clonal origin from other blood cell types. We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell.


Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Células Clonais/citologia , Células Clonais/metabolismo , Análise de Sequência/métodos , Análise de Célula Única , Peixe-Zebra/anatomia & histologia , Nadadeiras de Animais/citologia , Animais , Encéfalo/citologia , Sistemas CRISPR-Cas/genética , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Olho/citologia , Feminino , Genes Reporter/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Especificidade de Órgãos , Regeneração , Transcriptoma , Imagem Corporal Total , Peixe-Zebra/embriologia , Peixe-Zebra/genética
18.
Arch. Soc. Esp. Oftalmol ; 93(3): 119-125, mar. 2018. ilus
Artigo em Espanhol | IBECS | ID: ibc-172244

RESUMO

Introducción: La enfermedad de Stargardt es la maculopatía más frecuente en la edad infantil y adulta. Presenta un origen genético por afectación principalmente del gen ABCA4 con herencia autosómica recesiva. Se trata de un gen con características especiales por su gran tamaño y comportamiento, mostrando una elevada tasa de mutaciones. La aparición, desarrollo y accesibilidad económica de las técnicas de secuenciación masiva permiten realizar el diagnóstico genético de la enfermedad de Stargardt. Pacientes y métodos: Se presentan 2 casos clínicos diagnosticados genéticamente de enfermedad de Stargardt mediante la realización de un panel de secuenciación masiva de 298 genes. Resultados: Los pacientes presentaban un fenotipo de maculopatía de ojo de buey con ausencia de flecks y las siguientes mutaciones: c.G5882A:p.Gly1961Glu y c.C3056T:p.T1019M para el caso 1; c.G5882A:p.Gly1961Glu y c.287del:p.Asn96Thrfs·19 para el caso 2. Ambos pacientes comparten la mutación c.G5882A:p.Gly1961Glu que explica su fenotipo similar característico. Conclusiones: La secuenciación masiva es especialmente útil en la enfermedad de Stargardt, pues el gen ABCA4 presenta un gran tamaño y elevada heterogeneidad polimórfica, que se traduce en una amplia variabilidad clínica (AU)


Introduction: Stargardt's disease is the most frequent form of inherited macular dystrophy in children and adults. It is a genetic eye disorder caused by mutations in ABCA4 gene with an autosomal recessive inheritance. ABCA4 is a very polymorphic and large gene containing 50 exons. The development of next generation sequencing (NGS) can be used for the genetic diagnosis of this disease. Patients and methods: A report is presented on two patients with a clinical diagnosis of Stargardt's disease whose genetic confirmation was performed by a NGS panel of 298 genes. Results: Clinically, the patients showed bull's eye maculopathy and absence of flecks, and genetically they shared the Gly1961Glu mutation that could explain their common phenotype, together with c.C3056T:p.T1019M for case 1, and c.287del:p.Asn96Thrfs·19 for case 2. Conclusions: NGS is particularly useful in the diagnosis of Stargardt's disease as ABCA4 is a large gene with a high allelic heterogeneity that causes a wide range of clinical manifestations (AU)


Assuntos
Humanos , Masculino , Feminino , Adulto Jovem , Adulto , Análise de Sequência/métodos , Distrofias Retinianas/genética , Testes Genéticos/métodos , Mutação/genética , Terapia Genética , Transplante de Células-Tronco
19.
Enferm. infecc. microbiol. clín. (Ed. impr.) ; 36(2): 91-94, feb. 2018. ilus, tab, graf
Artigo em Espanhol | IBECS | ID: ibc-170696

RESUMO

Objetivo: Generar una secuencia consenso a partir de los datos de secuenciación masiva obtenidos en estudios de resistencias a antiretrovirales, que sea representativa de la secuencia Sanger y que sirva para estudios de epidemiología molecular. Material y métodos: En 62 pacientes se obtuvo la secuencia de transcriptasa reversa-proteasa, mediante Sanger (Trugene-Siemens), y NGS (454GSJunior-Roche). Las secuencias consenso NGS se generaron con Mesquite, seleccionando umbrales 10%, 15% y 20%. Para el estudio filogenético se empleó MEGA. Resultados: Utilizando el umbral 10%, 17/62 pacientes presentaron secuencias pareadas NGS-Sanger, con una mediana de bootstrap del 88% (IQR83,5-95,5). La asociación aumenta a 36/62 pacientes y el bootstrap, a 94% (IQR85,5-98), y alcanza el máximo al 20% en 61/62 pacientes, bootstrap 99% (IQR98-100). Conclusión: Mostramos un método seguro para generar secuencias consenso NGS para su uso en estudios de epidemiología molecular procesadas con umbral 20%, de fácil uso y aplicación en los servicios de microbiología clínica (AU)


Objective: To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. Material and methods: Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. Results: At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). Conclusion: A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies (AU)


Assuntos
Humanos , Adulto , HIV , Infecções por HIV/epidemiologia , Análise de Sequência/métodos , Epidemiologia Molecular/métodos , Resistência a Medicamentos , Epidemiologia Molecular/estatística & dados numéricos , Inibidores da Transcriptase Reversa/análise , Inibidores da Transcriptase Reversa/isolamento & purificação , Filogenia , Antirretrovirais
20.
Carbohydr Polym ; 183: 81-90, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29352895

RESUMO

Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI-MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC-MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dalteparina/química , Oligossacarídeos/química , Análise de Sequência/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos
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