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1.
Adv Exp Med Biol ; 1168: 103-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31713167

RESUMO

The past two decades have seen unprecedented advances in the field of oncogenomics. The ongoing characterization of neoplastic tissues through genomic techniques has transformed many aspects of cancer research, diagnosis, and treatment. However, identifying sequence variants with biological and clinical significance is a challenging endeavor. In order to accomplish this task, variants must be annotated and interpreted using various online resources. Data on protein structure, functional prediction, variant frequency in relevant populations, and multiple other factors have been compiled in useful databases for this purpose. Thus, understanding the available online resources for the annotation and interpretation of sequence variants is critical to aid molecular pathologists and researchers working in this space.


Assuntos
Bases de Dados Genéticas , Privacidade Genética , Neoplasias , Farmacogenética , Privacidade Genética/tendências , Variação Genética , Recursos em Saúde , Humanos , Internet , Neoplasias/fisiopatologia , Neoplasias/terapia , Análise de Sequência de DNA/normas , Análise de Sequência de DNA/tendências
2.
BMC Bioinformatics ; 20(1): 474, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521109

RESUMO

BACKGROUND: In most mammals, a vast array of genes coding for chemosensory receptors mediates olfaction. Odorant receptor (OR) genes generally constitute the largest multifamily (> 1100 intact members in the mouse). From the whole pool, each olfactory neuron expresses a single OR allele following poorly characterized mechanisms termed OR gene choice. OR genes are found in genomic aggregations known as clusters. Nearby enhancers, named elements, are crucial regulators of OR gene choice. Despite their importance, searching for new elements is burdensome. Other chemosensory receptor genes responsible for smell adhere to expression modalities resembling OR gene choice, and are arranged in genomic clusters - often with chromosomal linkage to OR genes. Still, no elements are known for them. RESULTS: Here we present an inexpensive framework aimed at predicting elements. We redefine cluster identity by focusing on multiple receptor gene families at once, and exemplify thirty - not necessarily OR-exclusive - novel candidate enhancers. CONCLUSIONS: The pipeline we introduce could guide future in vivo work aimed at discovering/validating new elements. In addition, our study provides an updated and comprehensive classification of all genomic loci responsible for the transduction of olfactory signals in mammals.


Assuntos
Algoritmos , Elementos Facilitadores Genéticos , Genômica/métodos , Receptores Odorantes/genética , Análise de Sequência de DNA/normas , Animais , Humanos , Camundongos , Ratos
3.
Hum Genet ; 138(7): 757-769, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31168775

RESUMO

An ethnicity is characterized by genomic fragments, single nucleotide polymorphisms (SNPs), and structural variations specific to it. However, the widely used 'standard human reference genome' GRCh37/38 is based on Caucasians. Therefore, de novo-assembled reference genomes for specific ethnicities would have advantages for genetics and precision medicine applications, especially with the long-read sequencing techniques that facilitate genome assembly. In this study, we assessed the de novo-assembled Chinese Han reference genome HX1 vis-à-vis the standard GRCh38 for improving the quality of assembly and for ethnicity-specific applications. Surprisingly, all genomic sequencing datasets mapped better to GRCh38 than to HX1, even for the datasets of the Chinese Han population. This gap was mainly due to the massive structural misassembly of the HX1 reference genome rather than the SNPs between the ethnicities, and this misassembly could not be corrected by short-read whole-genome sequencing (WGS). For example, HX1 and the other de novo-assembled personal genomes failed to assemble the mitochondrial genome as a contig. We mapped 97.1% of dbSNP, 98.8% of ClinVar, and 97.2% of COSMIC variants to HX1. HX1-absent, non-synonymous ClinVar SNPs were involved in 140 genes and many important functions in various diseases, most of which were due to the assembly failure of essential exons. In contrast, the HX1-specific regions were scantly expressible, as shown in the cell lines and clinical samples of Chinese patients. Our results demonstrated that the de novo-assembled individual genome such as HX1 did not have advantages against the standard GRCh38 genome due to insufficient assembly quality, and that it is, therefore, not recommended for common use.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Grupos Étnicos/genética , Genoma Humano , Genômica/normas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Padrões de Referência , Análise de Sequência de DNA/normas , Algoritmos , Mapeamento de Sequências Contíguas , Genética Populacional , Humanos , Polimorfismo de Nucleotídeo Único , Transcriptoma
4.
Western Pac Surveill Response J ; 10(1): 32-38, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31110840

RESUMO

Introduction: There are two methods of reverse transcription polymerase chain reaction (RT-PCR) that have been the common methods to detect influenza infections: conventional and real-time RT-PCR. From December 2017 to March 2018, several missed diagnoses of influenza A(H1)pdm09 using real-time RT-PCR were reported in northern Viet Nam. This study investigated how these missed detections occurred to determine their effect on the surveillance of influenza. Methods: The haemagglutinin (HA) segments of A(H1N1)pdm09 from both real-time RT-PCR positive and negative samples were isolated and sequenced. The primer and probe sets in the HA gene were checked for mismatches, and phylogenetic analyses were performed to determine the molecular epidemiology of these viruses. Results: There were 86 positive influenza A samples; 32 were A(H1)pdm09 positive by conventional RT-PCR but were negative by real-time RT-PCR. Sequencing was conducted on 23 influenza (H1N1)pdm09 isolates that were recovered from positive samples. Eight of these were negative for A(H1)pdm09 by real-time RT-PCR. There were two different mismatches in the probe target sites of the HA gene sequences of all isolates (n = 23) with additional mismatches only at position 7 (template binding site) identified for all eight negative real-time RT-PCR isolates. The prime target sites had no mismatches. Phylogenetic analysis of the HA gene showed that both the positive and negative real-time RT-PCR isolates were grouped in clade 6B.1; however, the real-time RT-PCR negative viruses were located in a subgroup that referred to substitution I295V. Conclusion: Constant monitoring of genetic changes in the circulating influenza A(H1N1)pdm09 viruses is important for maintaining the sensitivity of molecular detection assays.


Assuntos
Diagnóstico Tardio/tendências , Influenza Humana/diagnóstico , Análise de Sequência de DNA/normas , Testes de Hemaglutinação/métodos , Hemaglutininas/análise , Hemaglutininas/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/epidemiologia , Influenza Humana/mortalidade , Mutação/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/tendências , Vietnã
5.
Artif Cells Nanomed Biotechnol ; 47(1): 636-643, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30873882

RESUMO

Presently, clopidogrel is the standard therapeutic drugs for antiplatelet therapy. Variants in the CYP2C19 gene influence the clinical response of clopidogrel. Thus, the US Food and Drug Administration suggested CYP2C19 genotyping needs to identify before taking medicine. Due to high cost, time consuming, and sophisticated instruments, current single nucleotide polymorphism detection methods are limited in clinical application. In the present study, we established a genotyping method for CYP2C19, which combines amplification refractory mutation system (ARMS)-PCR with a lateral flow assay used gold magnetic nanoparticles (GMNPs) named as PCR-gold magnetic lateral flow assay system (PCR-GoldMag LFA). The PCR products with specific genotype can be explained within 5 minutes, either through visual or by a magnetic reader automatically according to the captured GMNPs probes on the test lines of the LFA strips. The limit of detection of this method is 5 ng of genomic DNA. The PCR-GoldMag LFA system was applied in a clinical trial with 1356 samples of Han Chinese. The concordance rate between the LFA system and sequencing is 99.93%. The allele frequency of CYP2C19*2 and CYP2C19*3 are 30.38 and 7.08% in Han Chinese, respectively. This method provides a new way in the clinical application of CYP2C19 genotyping to guide the clopidogrel medication.


Assuntos
Citocromo P-450 CYP2C19/genética , Técnicas de Genotipagem/instrumentação , Ouro/química , Nanopartículas de Magnetita/química , Reação em Cadeia da Polimerase , Citocromo P-450 CYP2C19/sangue , Frequência do Gene , Genótipo , Técnicas de Genotipagem/economia , Humanos , Mutação , Reação em Cadeia da Polimerase/economia , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas , Fatores de Tempo
6.
Genome Biol ; 20(1): 50, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30867008

RESUMO

BACKGROUND: Sequencing errors are key confounding factors for detecting low-frequency genetic variants that are important for cancer molecular diagnosis, treatment, and surveillance using deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, PCR enrichment, and sequencing. In this study, we use current NGS technology to systematically investigate these questions. RESULTS: By evaluating read-specific error distributions, we discover that the substitution error rate can be computationally suppressed to 10-5 to 10-4, which is 10- to 100-fold lower than generally considered achievable (10-3) in the current literature. We then quantify substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets. We find that error rates differ by nucleotide substitution types, ranging from 10-5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10-4 for A>G/T>C changes. Furthermore, C>T/G>A errors exhibit strong sequence context dependency, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. We also find that more than 70% of hotspot variants can be detected at 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression. CONCLUSIONS: We present the first comprehensive analysis of sequencing error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS analysis accuracy both experimentally and computationally, ultimately enhancing the precision of deep sequencing.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/normas , Neoplasias/genética , Reação em Cadeia da Polimerase/normas , Análise de Sequência de DNA/normas , Software , Estudos de Casos e Controles , Humanos , Mutação , Controle de Qualidade
7.
Workplace Health Saf ; 67(6): 268-274, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30924742

RESUMO

Occupational health nurses play a key role in evaluating innovative technologies that can aid in providing safe and rapid care and reduce lost work time. A nurse-led employee health clinic participated in a validation study of a novel pathogen detection technique developed by GeneCapture, Inc. Their proposed portable urinary tract infection (UTI) in vitro diagnostic test was challenged with discarded, deidentified urine samples from patients presenting with typical UTI symptoms collected at two university clinics and two multiphysician practices. GeneCapture's panel for this study was designed to rapidly identify the genetic signature of seven organisms: gram-negative Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa; gram-positive Enterococcus faecalis and Staphylococcus aureus; and fungal Candida species. The results from 40 clinical samples were in 95% agreement (90% specificity, 100% sensitivity) with traditional urine culture results from routine analysis. This successful occupational health nursing collaboration and validation study shows promise for point-of-care diagnoses and earlier treatment for workers with UTIs.


Assuntos
Enfermagem do Trabalho/métodos , Análise de Sequência de DNA/normas , Infecções Urinárias/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Invenções/tendências , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Masculino , Pessoa de Meia-Idade , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Análise de Sequência de DNA/métodos , Serviços de Saúde para Estudantes/organização & administração , Estudos de Validação como Assunto
8.
Methods Mol Biol ; 1963: 195-213, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30875055

RESUMO

Recent methodological advances have transformed the field of ancient DNA (aDNA). Basic bioinformatics skills are becoming essential requirements to process and analyze the sheer amounts of data generated by current aDNA studies and in biomedical research in general. This chapter is intended as a practical guide to the assembly of ancient mitochondrial genomes, directly from genomic DNA-derived next-generation sequencing (NGS) data, specifically in the absence of closely related reference genomes. In a hands-on tutorial suitable for readers with little to no prior bioinformatics experience, we reconstruct the mitochondrial genome of a woolly mammoth deposited ~45,000 years ago. We introduce key software tools and outline general strategies for mitogenome assembly, including the critical quality assessment of assembly results without a reference genome.


Assuntos
DNA Antigo/análise , DNA Mitocondrial/genética , Genoma Mitocondrial , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/normas , Humanos , Padrões de Referência , Análise de Sequência de DNA/métodos , Software
9.
Eur J Clin Microbiol Infect Dis ; 38(6): 1059-1070, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30834996

RESUMO

Recent advancements in next-generation sequencing (NGS) have provided the foundation for modern studies into the composition of microbial communities. The use of these NGS methods allows for the detection and identification of ('difficult-to-culture') microorganisms using a culture-independent strategy. In the field of routine clinical diagnostics however, the application of NGS is currently limited to microbial strain typing for epidemiological purposes only, even though the implementation of NGS for microbial community analysis may yield clinically important information. This lack of NGS implementation is due to many different factors, including issues relating to NGS method standardization and result reproducibility. In this review article, the authors provide a general introduction to the most widely used NGS methods currently available (i.e., targeted amplicon sequencing and shotgun metagenomics) and the strengths and weaknesses of each method is discussed. The focus of the publication then shifts toward 16S rRNA gene NGS methods, which are currently the most cost-effective and widely used NGS methods for research purposes, and are therefore more likely to be successfully implemented into routine clinical diagnostics in the short term. In this respect, the experimental pitfalls and biases created at each step of the 16S rRNA gene NGS workflow are explained, as well as their potential solutions. Finally, a novel diagnostic microbiota profiling platform ('MYcrobiota') is introduced, which was developed by the authors by taking into consideration the pitfalls, biases, and solutions explained in this article. The development of the MYcrobiota, and future NGS methodologies, will help pave the way toward the successful implementation of NGS methodologies into routine clinical diagnostics.


Assuntos
Testes Diagnósticos de Rotina/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , Infecção/diagnóstico , Microbiota/genética , DNA Bacteriano/genética , DNA Bacteriano/normas , Humanos , Infecção/epidemiologia , Infecção/microbiologia , Metagenômica/normas , Técnicas Microbiológicas/normas , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/normas , Análise de Sequência de DNA/normas
10.
Int J Syst Evol Microbiol ; 69(4): 895-898, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30832757

RESUMO

The World Federation of Culture Collections and the World Data Center for Microorganisms (wdcm) initiated an international community-led project to sequence and annotate newly described prokaryotic taxa. This sequencing project aims to cooperate with international culture collections and the International Journal of Systematic and Evolutionary Microbiology and contribute to the expansion of whole genome sequencing databases for type strains. It will provide global microbial taxonomists with free standard genome sequencing and annotation services. Taxonomists are encouraged to contact the wdcm and participant culture collections to submit a type strain sequencing proposal.


Assuntos
Classificação/métodos , Bases de Dados Genéticas , Genômica/normas , Células Procarióticas/classificação , Análise de Sequência de DNA/normas
11.
Nat Commun ; 10(1): 977, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816127

RESUMO

The advent of next-generation sequencing (NGS) has accelerated biomedical research by enabling the high-throughput analysis of DNA sequences at a very low cost. However, NGS has limitations in detecting rare-frequency variants (< 1%) because of high sequencing errors (> 0.1~1%). NGS errors could be filtered out using molecular barcodes, by comparing read replicates among those with the same barcodes. Accordingly, these barcoding methods require redundant reads of non-target sequences, resulting in high sequencing cost. Here, we present a cost-effective NGS error validation method in a barcode-free manner. By physically extracting and individually amplifying the DNA clones of erroneous reads, we distinguish true variants of frequency > 0.003% from the systematic NGS error and selectively validate NGS error after NGS. We achieve a PCR-induced error rate of 2.5×10-6 per base per doubling event, using 10 times less sequencing reads compared to those from previous studies.


Assuntos
Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Clonagem Molecular , Código de Barras de DNA Taxonômico , DNA Bacteriano/genética , Escherichia coli/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/normas , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Reação em Cadeia da Polimerase , Controle de Qualidade , Análise de Sequência de DNA/normas , Análise de Sequência de DNA/estatística & dados numéricos
12.
Methods Mol Biol ; 1963: 1-13, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30875038

RESUMO

Entering into the world of ancient DNA research is nontrivial. Because the DNA in most ancient specimens is degraded to some extent, the potential is high for contamination of ancient samples, ancient DNA extracts, and genomic sequencing libraries prepared from these extracts with non-degraded DNA from the present-day environment. To minimize the risk of contamination in ancient DNA environments, experimental protocols specific to handling ancient specimens, including those that outline the design and layout of laboratory space, have been introduced. Here, we outline challenges associated with working with ancient samples, including providing guidelines for setting up a new ancient DNA laboratory. We also discuss steps that can be taken at the sample collection and preparation stage to minimize the potential for contamination of ancient DNA experiments with exogenous sources of DNA.


Assuntos
Contaminação por DNA , DNA Antigo/análise , Análise de Sequência de DNA/métodos , Animais , Dano ao DNA , DNA Antigo/química , DNA Antigo/isolamento & purificação , Fósseis , Humanos , Laboratórios/normas , Análise de Sequência de DNA/normas
13.
Genome Res ; 29(4): 646-656, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30846530

RESUMO

We report on the development of a methylation analysis workflow for optical detection of fluorescent methylation profiles along chromosomal DNA molecules. In combination with Bionano Genomics genome mapping technology, these profiles provide a hybrid genetic/epigenetic genome-wide map composed of DNA molecules spanning hundreds of kilobase pairs. The method provides kilobase pair-scale genomic methylation patterns comparable to whole-genome bisulfite sequencing (WGBS) along genes and regulatory elements. These long single-molecule reads allow for methylation variation calling and analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell second-generation sequencing. The method is applied here to study facioscapulohumeral muscular dystrophy (FSHD), simultaneously recording the haplotype, copy number, and methylation status of the disease-associated, highly repetitive locus on Chromosome 4q.


Assuntos
Metilação de DNA , Análise de Sequência de DNA/métodos , Variação Genética , Humanos , Distrofia Muscular Facioescapuloumeral/genética , Análise de Sequência de DNA/normas
14.
BMC Res Notes ; 12(1): 159, 2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902062

RESUMO

OBJECTIVE: Stabilising samples of microbial communities for DNA extraction without access to laboratory equipment can be a challenging task. In this paper we propose a method using filter paper disks for the preservation of DNA from diverse microbial communities which are found in a fermented milk product. RESULTS: Small adaptations to the DNA extraction method used for liquid fermented milk delivered DNA of sufficient amounts and quality to be used for later analyses, e.g. full community 16S amplicon sequencing. The microbial community structure obtained via the filter paper method showed sufficient resemblance to the structure obtain via the traditional DNA extraction from the liquid milk sample. This method can therefore successfully be used to analyse diverse microbial communities from fermented milk products from remote areas.


Assuntos
Produtos Fermentados do Leite/microbiologia , DNA Bacteriano/isolamento & purificação , Microbiologia de Alimentos/métodos , Microbiota , Preservação Biológica/métodos , Análise de Sequência de DNA/métodos , Manejo de Espécimes/métodos , Microbiologia de Alimentos/normas , Preservação Biológica/normas , Análise de Sequência de DNA/normas , Manejo de Espécimes/normas , Zâmbia
15.
Mol Genet Genomic Med ; 7(3): e545, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30706702

RESUMO

BACKGROUND: We evaluated the performance of a cell-free DNA (cfDNA) prenatal screening assay for trisomies 21, 18, and 13, and sex chromosome aneuploidies (SCAs) among a population of pregnant women that included both those at average and high risk. METHODS: Specimen collection, cfDNA extraction, massively parallel sequencing, and bioinformatics analysis were conducted per laboratory protocol. Assay results, concordance with pregnancy outcomes, and performance characteristics were evaluated. RESULTS: A total 75,658 specimens from 72,176 individual pregnant women were received. Technical reasons accounted for 288 (0.4% of all received samples) tests not performed. In the final analysis cohort (N = 69,794), 13% of pregnancies were considered at average risk and 87% at high risk. Mean gestational age at specimen collection was 15.1 weeks. Of the 69,794 unique pregnancies, 1,359 (1.9%) had positive test results. Among the results with confirmed outcomes, PPV for trisomies 21, 18, and 13 was 98.1%, 88.2%, and 59.3%, respectively; the PPV was 69.0% for SCAs and 75.0% for microdeletions. Overall, PPV was 87.2%, sensitivity was 97.9%, and specificity was 99.9%. CONCLUSION: This cfDNA prenatal screening assay provides highly accurate discrimination between affected and unaffected pregnancies among a population of pregnant women at average and high risk for fetal genetic abnormalities.


Assuntos
Aneuploidia , Ácidos Nucleicos Livres/genética , Transtornos Cromossômicos/diagnóstico , Testes Genéticos/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Ácidos Nucleicos Livres/química , Transtornos Cromossômicos/genética , Feminino , Testes Genéticos/normas , Humanos , Gravidez , Diagnóstico Pré-Natal/normas , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
16.
Mol Genet Genomic Med ; 7(4): e00597, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30767419

RESUMO

BACKGROUND: The noninvasive prenatal testing (NIPT) has been successfully used in the clinical screening of fetal trisomy 13, 18, and 21 in the last few years and researches on detecting sub-chromosomal copy number variations (CNVs) and monogenic diseases are also in progress. To date, multiple tests are needed in order to complete a full set of fetus disorder screening, which is costly and time consuming. Therefore, an integrated 3-in-1 NIPT approach will be in great demand by routine clinical practice in the near future. METHODS: We designed a target capture sequencing panel with an associate bioinformatics pipeline to create a novel multi-functional NIPT method and we evaluated its performance by testing 22 clinical samples containing aneuploidy, CNV, and single-gene disorder. Chromosomal aneuploidy and CNV were detected based on the Z-value approach, whereas single-gene disorder was identified by using the "pseudo-tetraploid" model to estimate the best-suited genotype for each locus. RESULTS: The performance of this newly constructed 3-in-1 system was promising. We achieved a 100% detection rate for chromosomal aneuploidies (7/7), a 100% diagnosis rate for fetus CNVs larger than 20 Mb (3/3), and an 86.4% accuracy for single-gene disorder screening (19/22). CONCLUSION: For the first time, we showed that it is possible to use just a single NIPT test to detect three distinct types of fetus disorder and laid a foundation for developing a cheaper, faster, and multi-functional NIPT method in the future.


Assuntos
Aneuploidia , Variações do Número de Cópias de DNA , Testes Genéticos/métodos , Mutação , Diagnóstico Pré-Natal/métodos , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Feminino , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Testes Genéticos/normas , Humanos , Projetos Piloto , Gravidez , Diagnóstico Pré-Natal/normas , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
18.
Mol Genet Genomic Med ; 7(3): e564, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30637984

RESUMO

BACKGROUND: Mutations in GBA cause Gaucher disease when biallelic and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA using long reads. METHODS: We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb amplicon from 102 individuals, including patients with Parkinson's and Gaucher diseases. We used NanoOK for quality metrics, NGMLR to align data (after comparing with GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing. RESULTS: We detected all known missense mutations in these samples, including the common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demonstrated the ability to phase mutations, confirm compound heterozygosity, and assign haplotypes. We also detected two known risk variants in some Parkinson's patients. Rare false positives were easily identified and filtered, with the Nanopolish quality score adjusted for the number of reads a very robust discriminator. In two individuals carrying a recombinant allele, we were able to detect and fully define it in one carrier, where it included a 55-base pair deletion, but not in another one, suggesting a limitation of the PCR enrichment method. Missense mutations were detected at the correct zygosity, except for the case where the RecNciI one was missed. CONCLUSION: The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants.


Assuntos
Doença de Gaucher/genética , Testes Genéticos/métodos , Glucosilceramidase/genética , Mutação de Sentido Incorreto , Análise de Sequência de DNA/métodos , Doença de Gaucher/diagnóstico , Testes Genéticos/normas , Humanos , Kit de Reagentes para Diagnóstico/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas
19.
Genes (Basel) ; 10(1)2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646604

RESUMO

The advent of third-generation sequencing (TGS) technologies, such as the Pacific Biosciences (PacBio) and Oxford Nanopore machines, provides new possibilities for contig assembly, scaffolding, and high-performance computing in bioinformatics due to its long reads. However, the high error rate and poor quality of TGS reads provide new challenges for accurate genome assembly and long-read alignment. Efficient processing methods are in need to prioritize high-quality reads for improving the results of error correction and assembly. In this study, we proposed a novel Read Quality Evaluation and Selection Tool (REQUEST) for evaluating the quality of third-generation long reads. REQUEST generates training data of high-quality and low-quality reads which are characterized by their nucleotide combinations. A linear regression model was built to score the quality of reads. The method was tested on three datasets of different species. The results showed that the top-scored reads prioritized by REQUEST achieved higher alignment accuracies. The contig assembly results based on the top-scored reads also outperformed conventional approaches that use all reads. REQUEST is able to distinguish high-quality reads from low-quality ones without using reference genomes, making it a promising alternative sequence-quality evaluation method to alignment-based algorithms.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/normas , Controle de Qualidade , Análise de Sequência de DNA/normas , Software , Animais , Drosophila melanogaster , Escherichia coli , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Yersinia pestis
20.
Genes (Basel) ; 10(1)2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30669388

RESUMO

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.


Assuntos
Anopheles/genética , Genoma de Inseto , Análise de Sequência de DNA/métodos , Animais , Mapeamento de Sequências Contíguas/métodos , Mapeamento de Sequências Contíguas/normas , Ploidias , Polimorfismo Genético , Análise de Sequência de DNA/normas
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