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1.
Nat Commun ; 10(1): 2163, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092822

RESUMO

Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis.


Assuntos
Neoplasias da Mama/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Linhagem Celular , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Camundongos , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
2.
Nat Commun ; 10(1): 1869, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015479

RESUMO

Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. Here we evaluate the potential of the Oxford Nanopore Technologies (ONT) MinION long-read sequencer for routine WGS by sequencing the reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome and severe immune dysregulation. We develop and apply a novel reference panel-free analytical method to infer and then exploit phase information which improves single-nucleotide variant (SNV) calling performance from otherwise modest levels. In the clinical sample, we identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they lie on the same paternal haplotype. Whilst consensus SNV-calling error rates from ONT data remain substantially higher than those from short-read methods, we demonstrate the substantial benefits of analytical innovation. Ongoing improvements to base-calling and SNV-calling methodology must continue for nanopore sequencing to establish itself as a primary method for clinical WGS.


Assuntos
Testes Genéticos/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanoporos , Sequenciamento Completo do Genoma/métodos , Adulto , Ataxia Cerebelar/diagnóstico , Ataxia Cerebelar/genética , Feminino , Genoma Humano/genética , Genômica/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Lactente , Masculino , Nanotecnologia , Pancitopenia/diagnóstico , Pancitopenia/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Supressoras de Tumor/genética , Sequenciamento Completo do Genoma/instrumentação
3.
Methods Mol Biol ; 1979: 73-85, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028633

RESUMO

Drop-Seq is a low-cost, high-throughput platform to profile thousands of cells by encapsualting them into individual droplets. Uniquely barcoded mRNA capture microparticles and cells are coconfined through a microfluidic device within the droplets where they undergo cell lysis and RNA hybridiztion. After breaking the droplets and pooling the hybridized particles, reverse transcription, PCR, and sequencing in single reactions allow to generate data from thousands of single-cell transcriptomes while maintaining information on the cellular origin of each transcript.


Assuntos
Perfilação da Expressão Gênica/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Dispositivos Lab-On-A-Chip , Análise de Célula Única/instrumentação , Animais , Desenho de Equipamento , Perfilação da Expressão Gênica/economia , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Dispositivos Lab-On-A-Chip/economia , Análise de Célula Única/economia , Análise de Célula Única/métodos , Transcriptoma
4.
Methods Mol Biol ; 1979: 111-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028635

RESUMO

Seq-Well is a low-cost picowell platform that can be used to simultaneously profile the transcriptomes of thousands of cells from diverse, low input clinical samples. In Seq-Well, uniquely barcoded mRNA capture beads and cells are co-confined in picowells that are sealed using a semipermeable membrane, enabling efficient cell lysis and mRNA capture. The beads are subsequently removed and processed in parallel for sequencing, with each transcript's cell of origin determined via the unique barcodes. Due to its simplicity and portability, Seq-Well can be performed almost anywhere.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Mensageiro/genética , Análise de Célula Única/métodos , Animais , Desenho de Equipamento , Perfilação da Expressão Gênica/economia , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Membranas Artificiais , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Transcrição Reversa , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Análise de Célula Única/economia , Análise de Célula Única/instrumentação
5.
Methods Mol Biol ; 1979: 155-176, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028637

RESUMO

Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection. However, due to the complexity of PBMCs, which contain multiple different cell types, studying the function of the individual cell types can be difficult, and often studies rely on bulk measurements. Here, we describe the analysis of PBMCs using single-cell RNA-sequencing in droplets. Data from these studies allow for the identification and quantification of the subpopulation of cells that make up the PBMC sample. In addition, differential gene expression between cell types and samples can be assessed.


Assuntos
Leucócitos Mononucleares/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Contagem de Células/métodos , Separação Celular/métodos , DNA Complementar/genética , Desenho de Equipamento , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucócitos Mononucleares/citologia , Receptores de Lipopolissacarídeos/análise , Monócitos/citologia , Monócitos/metabolismo , Transcrição Reversa , Análise de Sequência de RNA/instrumentação , Análise de Célula Única/instrumentação , Fluxo de Trabalho
6.
Nat Rev Genet ; 20(6): 356-370, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30886350

RESUMO

Antimicrobial resistance extracts high morbidity, mortality and economic costs yearly by rendering bacteria immune to antibiotics. Identifying and understanding antimicrobial resistance are imperative for clinical practice to treat resistant infections and for public health efforts to limit the spread of resistance. Technologies such as next-generation sequencing are expanding our abilities to detect and study antimicrobial resistance. This Review provides a detailed overview of antimicrobial resistance identification and characterization methods, from traditional antimicrobial susceptibility testing to recent deep-learning methods. We focus on sequencing-based resistance discovery and discuss tools and databases used in antimicrobial resistance studies.


Assuntos
Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/estatística & dados numéricos , Sequenciamento Completo do Genoma/métodos , Antibacterianos/farmacologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Aprendizado de Máquina , Metagenômica , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/instrumentação
7.
Methods Mol Biol ; 1922: 407-452, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838594

RESUMO

Rare genetic disorders are often challenging to diagnose. Anomalies of tooth number, shape, size, mineralized tissue structure, eruption, and resorption may exist as isolated symptoms or diseases but are often part of the clinical synopsis of numerous syndromes (Bloch-Zupan A, Sedano H, Scully C. Dento/oro/craniofacial anomalies and genetics, 1st edn. Elsevier, Boston, MA, 2012). Concerning amelogenesis imperfecta (AI), for example, mutations in a number of genes have been reported to cause isolated AI, including AMELX, ENAM, KLK4, MMP20, FAM83H, WDR72, C4orf26, SLC24A4, and LAMB3. In addition, many other genes such as DLX3, CNNM4, ROGDI, FAM20A, STIM1, ORAI1, and LTBP3 have been shown to be involved in developmental syndromes with enamel defects. The clinical presentation of the enamel phenotype (hypoplastic, hypomineralized, hypomature, or a combination of severities) alone does not allow a reliable prediction of possible causative genetic mutations. Understanding the potential genetic cause(s) of rare diseases is critical for overall health management of affected patient. One effective strategy to reach a genetic diagnosis is to sequence a selected gene panel chosen for a determined range of phenotypes. Here we describe a laboratory protocol to set up a specific gene panel for orodental diseases.


Assuntos
Anormalidades Craniofaciais/genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças Raras/genética , Anormalidades Dentárias/genética , Amelogênese Imperfeita/diagnóstico , Amelogênese Imperfeita/genética , Anormalidades Craniofaciais/diagnóstico , DNA/genética , Desenho de Equipamento , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Doenças Raras/diagnóstico , Anormalidades Dentárias/diagnóstico
8.
Mol Cell ; 73(5): 1075-1082.e4, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849388

RESUMO

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.


Assuntos
Anticorpos/metabolismo , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligopeptídeos/metabolismo , Análise Serial de Proteínas/métodos , Afinidade de Anticorpos , Especificidade de Anticorpos , Automação Laboratorial , Sítios de Ligação de Anticorpos , Catálise , Análise Mutacional de DNA/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Cinética , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligopeptídeos/genética , Análise Serial de Proteínas/instrumentação , Ligação Proteica , Engenharia de Proteínas , Fluxo de Trabalho
9.
Microbiome ; 7(1): 44, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898140

RESUMO

BACKGROUND: Wastewater treatment plants (WWTPs) are recognized as hotspots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). Despite our understanding of the composition and distribution of ARGs in WWTPs, the genetic location, host, and fate of ARGs remain largely unknown. RESULTS: In this study, we combined Oxford Nanopore and Illumina metagenomics sequencing to comprehensively uncover the resistome context of influent, activated sludge, and effluent of three WWTPs and simultaneously track the hosts of the ARGs. The results showed that most of the ARGs detected in all compartments of the WWTPs were carried by plasmids. Transposons and integrons also showed higher prevalence on plasmids than on the ARG-carrying chromosome. Notably, integrative and conjugative elements (ICEs) carrying five types of ARGs were detected, and they may play an important role in facilitating the transfer of ARGs, particularly for tetracycline and macrolide-lincosamide-streptogramin (MLS). A broad spectrum of ARGs carried by plasmids (29 subtypes) and ICEs (4 subtypes) was persistent across the WWTPs. Host tracking showed a variety of antibiotic-resistant bacteria in the effluent, suggesting the high potential for their dissemination into receiving environments. Importantly, phenotype-genotype analysis confirmed the significant role of conjugative plasmids in facilitating the survival and persistence of multidrug-resistant bacteria in the WWTPs. At last, the consistency in the quantitative results for major ARGs types revealed by Nanopore and Illumina sequencing platforms demonstrated the feasibility of Nanopore sequencing for resistome quantification. CONCLUSION: Overall, these findings substantially expand our current knowledge of resistome in WWTPs, and help establish a baseline analysis framework to study ARGs in the environment.


Assuntos
Bactérias/classificação , Resistência Microbiana a Medicamentos , Metagenômica/instrumentação , Análise de Sequência de DNA/instrumentação , Águas Residuárias/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Transferência Genética Horizontal , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Lincosamidas/farmacologia , Macrolídeos/farmacologia , Metagenômica/métodos , Nanoporos , Filogenia , Análise de Sequência de DNA/métodos , Esgotos/microbiologia , Estreptograminas/farmacologia , Tetraciclina/farmacologia
10.
Nat Protoc ; 14(4): 1130-1168, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30903110

RESUMO

Among the different developed solid-state nanopores, nanopores constructed in a monolayer of molybdenum disulfide (MoS2) stand out as powerful devices for single-molecule analysis or osmotic power generation. Because the ionic current through a nanopore is inversely proportional to the thickness of the pore, ultrathin membranes have the advantage of providing relatively high ionic currents at very small pore sizes. This increases the signal generated during translocation of biomolecules and improves the nanopores' efficiency when used for desalination or reverse electrodialysis applications. The atomic thickness of MoS2 nanopores approaches the inter-base distance of DNA, creating a potential candidate for DNA sequencing. In terms of geometry, MoS2 nanopores have a well-defined vertical profile due to their atomic thickness, which eliminates any unwanted effects associated with uneven pore profiles observed in other materials. This protocol details all the necessary procedures for the fabrication of solid-state devices. We discuss different methods for transfer of monolayer MoS2, different approaches for the creation of nanopores, their applicability in detecting DNA translocations and the analysis of translocation data through open-source programming packages. We present anticipated results through the application of our nanopores in DNA translocations and osmotic power generation. The procedure comprises four parts: fabrication of devices (2-3 d), transfer of MoS2 and cleaning procedure (24 h), the creation of nanopores within MoS2 (30 min) and performing DNA translocations (2-3 h). We anticipate that our protocol will enable large-scale manufacturing of single-molecule-analysis devices as well as next-generation DNA sequencing.


Assuntos
Dissulfetos/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microtecnologia/métodos , Molibdênio/química , Nanoporos/ultraestrutura , Nanotecnologia/métodos , DNA/análise , DNA/genética , Diálise/instrumentação , Diálise/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Microtecnologia/instrumentação , Nanotecnologia/instrumentação , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos
11.
Microbiol Spectr ; 7(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30737915

RESUMO

In the decade and a half since the introduction of next-generation sequencing (NGS), the technical feasibility, cost, and overall utility of sequencing have changed dramatically, including applications for infectious disease epidemiology. Massively parallel sequencing technologies have decreased the cost of sequencing by more than 6 orders or magnitude over this time, with a corresponding increase in data generation and complexity. This review provides an overview of the basic principles, chemistry, and operational mechanics of current sequencing technologies, including both conventional Sanger and NGS approaches. As the generation of large amounts of sequence data becomes increasingly routine, the role of bioinformatics in data analysis and reporting becomes all the more critical, and the successful deployment of NGS in public health settings requires careful consideration of changing information technology, bioinformatics, workforce, and regulatory requirements. While there remain important challenges to the sustainable implementation of NGS in public health, in terms of both laboratory and bioinformatics capacity, the impact of these technologies on infectious disease surveillance and outbreak investigations has been nothing short of revolutionary. Understanding the important role that NGS plays in modern public health laboratory practice is critical, as is the need to ensure appropriate workforce, infrastructure, facilities, and funding consideration for routine NGS applications, future innovation, and rapidly scaling NGS-based infectious disease surveillance and outbreak response activities. *This article is part of a curated collection.


Assuntos
Biologia Computacional/métodos , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Análise de Dados , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Análise de Sequência de DNA/economia , Análise de Sequência de DNA/instrumentação
12.
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
13.
Int J Legal Med ; 133(3): 689-697, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30604102

RESUMO

Capillary electrophoresis (CE) is widely used in forensic genetics to study short tandem repeats (STRs). Recently, next-generation sequencing (NGS) platforms have facilitated the development of new strategies for forensic DNA typing. Several studies have shown that NGS successfully analyzes challenging samples. However, because NGS is complicated and time-consuming, it remains unclear whether NGS platforms offer significant advantages over CE for all forensic cases. Here, the MiSeq FGx system was used to test some cases that had previously been analyzed using CE. These cases included paternity test cases in which some samples exhibited locus inconsistencies; samples with off-ladder (OL) alleles; samples with triallelic patterns; and samples with amelogenin test abnormalities. The results generated by MiSeq FGx were compared to those previously generated by CE. The MiSeq FGx and CE results were consistent with the exception of three samples, where inconsistencies were observed at the Penta D locus. For all three incongruent samples, the MiSeq FGx results were correct. Sequence analysis indicated that, in two cases, mismatches were due to undetected alleles rather than mutations. In two additional cases, mutation sources were identified, and in a fifth case, mutation step size was reconsidered. MiSeq FGx was used to identify OL alleles and samples with amelogenin test abnormalities. For cases where verification was required via CE analysis, the simultaneous NGS amplification of several types of multiple genetic markers improved testing efficiency. In addition, we identified additional sequence variants at autosomal, Y chromosomal, and X chromosomal STR loci in the Han Chinese population from northern China. Our results will be useful for future forensic analyses of STR genotypes in Chinese populations. It is likely that NGS would be more widely used in forensic genetics if costs and procedure complexity were reduced.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Análise de Sequência de DNA , China , Cromossomos Humanos X , Cromossomos Humanos Y , Impressões Digitais de DNA , Eletroforese Capilar , Grupos Étnicos/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Repetições de Microssatélites , Mutação , Reação em Cadeia da Polimerase
14.
Methods Mol Biol ; 1912: 55-76, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30635890

RESUMO

Circular RNAs are an emerging class of transcript isoforms created by unique back splicing of exons to form a closed covalent circular structure. While initially considered as product of aberrant splicing, recent evidence suggests unique functions and conservation across evolution. While circular RNAs could be largely attributed to have little or no potential to encode for proteins, recent evidence points to at least a small subset of circular RNAs which encode for peptides. Circular RNAs are also increasingly shown to be biomarkers for a number of diseases including neurological disorders and cancer. The advent of deep sequencing has enabled large-scale identification of circular RNAs in human and other genomes. A number of computational approaches have come up in recent years to query circular RNAs on a genome-wide scale from RNA-seq data. In this chapter, we describe the application and methodology of identifying circular RNAs using three popular computational tools: FindCirc, Segemehl, and CIRI along with approaches for experimental validation of the unique splice junctions.


Assuntos
Biologia Computacional/métodos , Anotação de Sequência Molecular/métodos , Neoplasias/diagnóstico , Doenças do Sistema Nervoso/diagnóstico , RNA/isolamento & purificação , Biomarcadores , Biologia Computacional/instrumentação , Conjuntos de Dados como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias/genética , Doenças do Sistema Nervoso/genética , RNA/genética , RNA/metabolismo , Processamento de RNA , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Software
15.
Methods Mol Biol ; 1912: 77-110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30635891

RESUMO

Long noncoding RNAs (lncRNAs) belong to a class of RNA transcripts that do not have the potential to code for proteins. LncRNAs were largely discovered in the transcriptomes of human and several model organisms, using next-generation sequencing (NGS) approaches, which have enabled a comprehensive genome scale annotation of transcripts. LncRNAs are known to have dynamic expression status and have the potential to orchestrate gene regulation at the epigenetic, transcriptional, and posttranscriptional levels. Here we describe the experimental methods involved in the discovery of lncRNAs from the transcriptome of a popular model organism zebrafish (Danio rerio). A structured and well-designed computational analysis pipeline subsequent to the RNA sequencing can be instrumental in revealing the diversity of the lncRNA transcripts. We describe one such computational pipeline used for the discovery of novel lncRNA transcripts in zebrafish. We also detail the validation of the putative novel lncRNA transcripts using qualitative and quantitative assays in zebrafish.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Longo não Codificante/isolamento & purificação , Análise de Sequência de RNA/métodos , Animais , Biologia Computacional/instrumentação , Perfilação da Expressão Gênica/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Modelos Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Software , Transcriptoma/genética , Peixe-Zebra
16.
Methods Mol Biol ; 1912: 133-174, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30635893

RESUMO

Noncoding RNAs (ncRNAs) have received much attention due to their central role in gene expression and translational regulation as well as due to their involvement in several biological processes and disease development. Small noncoding RNAs (sncRNAs), such as microRNAs and piwiRNAs, have been thoroughly investigated and functionally characterized. Long noncoding RNAs (lncRNAs), known to play an important role in chromatin-interacting transcription regulation, posttranscriptional regulation, cell-to-cell signaling, and protein regulation, are also being investigated to further elucidate their functional roles.Next-generation sequencing (NGS) technologies have greatly aided in characterizing the ncRNAome. Moreover, the coupling of NGS technology together with bioinformatics tools has been essential to the genome-wide detection of RNA modifications in ncRNAs. RNA editing, a common human co-transcriptional and posttranscriptional modification, is a dynamic biological phenomenon able to alter the sequence and the structure of primary transcripts (both coding and noncoding RNAs) during the maturation process, consequently influencing the biogenesis, as well as the function, of ncRNAs. In particular, the dysregulation of the RNA editing machineries have been associated with the onset of human diseases.In this chapter we discuss the potential functions of ncRNA editing and describe the knowledge base and bioinformatics resources available to investigate such phenomenon.


Assuntos
Biologia Computacional/métodos , Regulação da Expressão Gênica , Edição de RNA , RNA não Traduzido/metabolismo , Animais , Biologia Computacional/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Análise de Sequência de RNA/métodos
17.
Methods Mol Biol ; 1912: 175-196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30635894

RESUMO

Proteins have a strong influence on the phenotype and their aberrant expression leads to diseases. MicroRNAs (miRNAs) are short RNA sequences which posttranscriptionally regulate protein expression. This regulation is driven by miRNAs acting as recognition sequences for their target mRNAs within a larger regulatory machinery. A miRNA can have many target mRNAs and an mRNA can be targeted by many miRNAs which makes it difficult to experimentally discover all miRNA-mRNA interactions. Therefore, computational methods have been developed for miRNA detection and miRNA target prediction. An abundance of available computational tools makes selection difficult. Additionally, interactions are not currently the focus of investigation although they more accurately define the regulation than pre-miRNA detection or target prediction could perform alone. We define an interaction including the miRNA source and the mRNA target. We present computational methods allowing the investigation of these interactions as well as how they can be used to extend regulatory pathways. Finally, we present a list of points that should be taken into account when investigating miRNA-mRNA interactions. In the future, this may lead to better understanding of functional interactions which may pave the way for disease marker discovery and design of miRNA-based drugs.


Assuntos
Biologia Computacional/métodos , Redes Reguladoras de Genes , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Animais , Biologia Computacional/instrumentação , Bases de Dados Genéticas , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Aprendizado de Máquina , MicroRNAs/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Software
18.
Methods Mol Biol ; 1912: 215-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30635896

RESUMO

microRNAs are evolutionarily conserved, endogenously produced, noncoding RNAs (ncRNAs) of approximately 19-24 nucleotides (nts) in length known to exhibit gene silencing of complementary target sequence. Their deregulated expression is reported in various disease conditions and thus has therapeutic implications. In the last decade, various computational resources are published in this field. In this chapter, we have reviewed bioinformatics resources, i.e., miRNA-centered databases, algorithms, and tools to predict miRNA targets. First section has enlisted more than 75 databases, which mainly covers information regarding miRNA registries, targets, disease associations, differential expression, interactions with other noncoding RNAs, and all-in-one resources. In the algorithms section, we have compiled about 140 algorithms from eight subcategories, viz. for the prediction of precursor (pre-) and mature miRNAs. These algorithms are developed on various sequence, structure, and thermodynamic based features incorporated into different machine learning techniques (MLTs). In addition, computational identification of miRNAs from high-throughput next generation sequencing (NGS) data and their variants, viz. isomiRs, differential expression, miR-SNPs, and functional annotation, are discussed. Prediction and analysis of miRNAs and their associated targets are also evaluated under miR-targets section providing knowledge regarding novel miRNA targets and complex host-pathogen interactions. In conclusion, we have provided comprehensive review of in silico resources published in miRNA research to help scientific community be updated and choose the appropriate tool according to their needs.


Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas/estatística & dados numéricos , MicroRNAs/metabolismo , Biologia Computacional/instrumentação , Redes Reguladoras de Genes , Inativação Gênica , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Interações Hospedeiro-Patógeno/genética , Humanos , Aprendizado de Máquina , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Análise de Sequência de RNA/estatística & dados numéricos
19.
PLoS One ; 14(1): e0206194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30629604

RESUMO

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Microquímica/métodos , Análise de Sequência de RNA/métodos , Automação Laboratorial , Redução de Custos , Estudos de Viabilidade , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Metagenômica/economia , Metagenômica/instrumentação , Microquímica/economia , Microquímica/instrumentação , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/instrumentação
20.
Methods Mol Biol ; 1858: 1-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30414106

RESUMO

As in any endeavor, the strategy applied to a genome project can mean the difference between success and failure. This is especially important when limited funding often means only a single approach may be tried at a given time. Although the advance of all areas of genomics and transcriptomics in recent years has led to an embarrassment of riches, methods in the field have not quite reached the turn-key production status for all species, despite being closer than ever. Here I contrast and compare the technical approaches to genome projects in the hope of enabling strategy choices with higher probabilities of success. Finally, I review the new technologies that are not yet widely distributed which are revolutionizing the future of genomics.


Assuntos
Artrópodes/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Animais , Artrópodes/classificação , Genoma de Inseto , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação
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