Name: Sequenciamento de Amplicon usando as tecnologias de sequenciamento de leitura longa (Vídeo) | JoVE Journal
Uploaded: 2025-08-29T13:40:00.000+00:00
Duration: 8 min 57 s
Description: Aprenda sequenciamento de amplicon com tecnologias de leitura longa para diagnóstico de tuberculose resistente a medicamentos. Este método oferece resultados precisos em ambientes com recursos limitados.

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<ol><li>Tuberculosis report. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Baltimore+Health+News%22">Baltimore Health News</a>. , WHO. (2022).</li>
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<li>Cabibbe, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+targeted+nextgeneration+sequencing+assay+on+a+portable+sequencing+platform+for+culturefree+detection+of+drugresistant+tuberculosis+from+clinical+samples%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Application of targeted nextgeneration sequencing assay on a portable sequencing platform for culturefree detection of drugresistant tuberculosis from clinical samples.</a> J Clin Microbiol. 58 (10), e0063220(2020).</li>
<li>Jouet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+amplicon+sequencing+for+culturefree+prediction+of+susceptibility+or+resistance+to+13+antituberculous+drugs%5BTitle%5D)+AND+%22Eur+Respir+J%22%5BJournal%5D)">Deep amplicon sequencing for culturefree prediction of susceptibility or resistance to 13 antituberculous drugs.</a> Eur Respir J. 57 (3), 2002338(2021).</li>
<li>NG, K. C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+well+do+routine+molecular+diagnostics+detect+Rifampin%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">How well do routine molecular diagnostics detect Rifampin.</a> J Clin Microbiol. 57 (1), e0128723(2019).</li>
<li>MacLean, E., Kohli, M., Weber, S. F., Suresh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+molecular+diagnosis+of+tuberculosis%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Advances in molecular diagnosis of tuberculosis.</a> J Clin Microbiol. 58 (10), e0158219(2020).</li>
<li>Sibandze, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+molecular+diagnostics+of+tuberculosis+resistance+by+targeted+stool+sequencing%5BTitle%5D)+AND+%22Genome+Med%22%5BJournal%5D)">Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing.</a> Genome Med. 14 (1), 42(2022).</li>
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<li>Hu, T., Chitnis, N., Monos, D., Dinh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nextgeneration+sequencing+technologies%3A+An+overview%5BTitle%5D)+AND+%22Hum+Immunol%22%5BJournal%5D)">Nextgeneration sequencing technologies: An overview.</a> Hum Immunol. 82 (10), 801-811 (2021).</li>
<li>Pervez, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+review+of+performance+of+nextgeneration+sequencing+platforms%5BTitle%5D)+AND+%22Biomed+Res+Int%22%5BJournal%5D)">A comprehensive review of performance of nextgeneration sequencing platforms.</a> Biomed Res Int. 2022, 3457806(2022).</li>
<li>Tafess, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targetedsequencing+workflows+for+comprehensive+drug+resistance+profiling+of+Mycobacterium+tuberculosis+cultures+using+two+commercial+sequencing+platforms%3A+Comparison+of+analytical+and+diagnostic+performance%2C+turnaround+time%2C+and+cost%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">Targetedsequencing workflows for comprehensive drug resistance profiling of Mycobacterium tuberculosis cultures using two commercial sequencing platforms: Comparison of analytical and diagnostic performance, turnaround time, and cost.</a> Clin Chem. 66 (6), 809-820 (2020).</li>
<li>Use of targeted nextgeneration sequencing to detect drugresistant tuberculosis. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22WHO+rapid+communication%22">WHO rapid communication</a>. , WHO. https://www.who.int/publications/i/item/9789240076372 (2023).</li>
<li>Hazra, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+wholegenome+sequencing+of+Mycobacterium+tuberculosis+on+treatment+outcomes+for+MDRTB%2FXDRTB%3A+A+systematic+review%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Impact of wholegenome sequencing of Mycobacterium tuberculosis on treatment outcomes for MDRTB/XDRTB: A systematic review.</a> Pharmaceutics. 15 (6), 1408(2023).</li>
<li>Shea, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+wholegenome+sequencing+and+reporting+of+drug+resistance+profiles+on+clinical+cases+of+Mycobacterium+tuberculosis+in+New+York+State%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Comprehensive wholegenome sequencing and reporting of drug resistance profiles on clinical cases of Mycobacterium tuberculosis in New York State.</a> J Clin Microbiol. 55 (6), 1871-1882 (2017).</li>
<li>Meehan, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+genome+sequencing+of+Mycobacterium+tuberculosis%3A+Current+standards+and+open+issues%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Whole genome sequencing of Mycobacterium tuberculosis: Current standards and open issues.</a> Nat Rev Microbiol. 17 (10), 533-545 (2019).</li>
<li>Heupink, T. H., Verboven, L., Warren, R. M., Van Rie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+and+accurate+genetic+variant+identification+from+contaminated+and+lowcoverage+Mycobacterium+tuberculosis+whole+genome+sequencing+data%5BTitle%5D)+AND+%22Microb+Genom%22%5BJournal%5D)">Comprehensive and accurate genetic variant identification from contaminated and lowcoverage Mycobacterium tuberculosis whole genome sequencing data.</a> Microb Genom. 7 (1), e000555(2021).</li>
<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interpretation+of+the+third+edition+of+WHO+consolidated+guidelines+on+tuberculosis%3A+Module+3%3A+Diagnosis%3A+Rapid+diagnostics+for+tuberculosis+detection%5BTitle%5D)+AND+%22Chin+J+Antituberc%22%5BJournal%5D)">Interpretation of the third edition of WHO consolidated guidelines on tuberculosis: Module 3: Diagnosis: Rapid diagnostics for tuberculosis detection.</a> Chin J Antituberc. 46 (2), 120-130 (2024).</li>
<li>Kambli, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+nextgeneration+sequencing+directly+from+sputum+for+comprehensive+genetic+information+on+drugresistant+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Tuberculosis%22%5BJournal%5D)">Targeted nextgeneration sequencing directly from sputum for comprehensive genetic information on drugresistant Mycobacterium tuberculosis.</a> Tuberculosis. 127 (1), 102051(2021).</li>
<li>Use of targeted nextgeneration sequencing to detect drugresistant tuberculosis. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Rapid+Communication%22">Rapid Communication</a>. , WHO. 1-8 (2023).</li>
<li>Nandlal, L., Perumal, R., Naidoo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+molecular+assays+for+the+diagnosis+of+drugresistant+tuberculosis%5BTitle%5D)+AND+%22Infect+Drug+Resist%22%5BJournal%5D)">Rapid molecular assays for the diagnosis of drugresistant tuberculosis.</a> Infect Drug Resist. 15 (1), 4971-4984 (2022).</li>
<li>Dippenaar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanopore+sequencing+for+Mycobacterium+tuberculosis%3A+A+critical+review+of+the+literature%2C+new+developments%2C+and+future+opportunities%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Nanopore sequencing for Mycobacterium tuberculosis: A critical review of the literature, new developments, and future opportunities.</a> J Clin Microbiol. 60 (5), e0123422(2022).</li>
<li>Chan, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+economical+drug+resistance+profiling+with+nanopore+MinION+for+clinical+specimens+with+low+bacillary+burden+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22BMC+Res+Notes%22%5BJournal%5D)">Rapid and economical drug resistance profiling with nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis.</a> BMC Res Notes. 13 (1), 123(2020).</li>
<li>Feuerriegel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+genomic+first+and+secondline+drug+resistance+prediction+from+clinical+Mycobacterium+tuberculosis+specimens+using+DeeplexMycTB%5BTitle%5D)+AND+%22Eur+Respir+J%22%5BJournal%5D)">Rapid genomic first and secondline drug resistance prediction from clinical Mycobacterium tuberculosis specimens using DeeplexMycTB.</a> Eur Respir J. 57 (4), 2004784(2021).</li>
<li>GenoScreen product brochure. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Deeplex+MycTB%22">Deeplex MycTB</a>. , GenoScreen. (2023).</li>
<li>Mtafya, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+approach+to+render+tuberculosis+samples+safe+for+application+of+tuberculosis+molecular+bacterial+load+assay+in+clinical+settings+without+a+biosafety+level+3+laboratory%5BTitle%5D)+AND+%22Tuberculosis%22%5BJournal%5D)">A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory.</a> Tuberculosis. 138 (1), 102194(2023).</li>
<li>Suganthi, P., Usharani, B., Venkateswari, R., Muthuraj, M. Biosafety practices in mycobacteriology laboratory. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSuganthi%2C+P+%22Research+Advances+in+Microbiology+and+Biotechnology%22">Research Advances in Microbiology and Biotechnology</a>. 3, B P International (a part of SCIENCEDOMAIN International). Chennai. 1-39 (2023).</li>
<li>consolidated guidelines on tuberculosis. Module 4: Treatment and care. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22WHO+guideline%22">WHO guideline</a>. , WHO. (2022).</li>
<li>Williams, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrixbased+DNA+extraction+for+targeted+nextgeneration+sequencing+on+decontaminated+sputum+samples%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Matrixbased DNA extraction for targeted nextgeneration sequencing on decontaminated sputum samples.</a> J Vis Exp. (181), e68147(2025).</li>
<li>Delahaye, C., Nicolas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequencing+DNA+with+nanopores%3A+Troubles+and+biases%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Sequencing DNA with nanopores: Troubles and biases.</a> PLoS One. 16 (2), e0245534(2021).</li>
<li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+identification+of+drugresistant+tuberculosis+genes+using+direct+PCR+amplification+and+Oxford+Nanopore+Technology+sequencing%5BTitle%5D)+AND+%22Can+J+Infect+Dis+Med+Microbiol%22%5BJournal%5D)">Rapid identification of drugresistant tuberculosis genes using direct PCR amplification and Oxford Nanopore Technology sequencing.</a> Can J Infect Dis Med Microbiol. 2022, 8845672(2022).</li>
<li>Tagliani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+and+nextgeneration+sequencingbased+drug+susceptibility+testing+unveil+high+levels+of+drugresistantTB+in+Djibouti%3A+Results+from+the+first+national+survey%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Culture and nextgeneration sequencingbased drug susceptibility testing unveil high levels of drugresistantTB in Djibouti: Results from the first national survey.</a> Sci Rep. 7 (1), 15076(2017).</li>
<li>Smith, C., Halse, T. A., Shea, J., Modestil, H., Fowler, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+nanopore+sequencing+for+clinical+diagnostics%3A+For+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Assessing nanopore sequencing for clinical diagnostics: For Mycobacterium tuberculosis.</a> J Clin Microbiol. 59 (7), e0123421(2021).</li>
</ol>

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sequenciamento de amplicon usando as tecnologias de sequenciamento de leitura longa

Amplicon Sequencing using the Long-Read Sequencing Technologies

Sequenciamento de Amplicon usando as tecnologias de sequenciamento de leitura longa

The World Health Organization (WHO) continues to emphasize the urgent need for a rapid, cost-effective, and user-friendly diagnostic method for ...

amplicon-sequencing-using-the-long-read-sequencing-technologies

Research

JoVE Journal

Biochemistry

Bioquímica

Aqui nós descrevemos um método para a preparação de ambos única leitura e emparelhado final Illumina mRNA Seq-libraries seqüenciamento para análise de expressão gênica com base na amplificação T7 RNA linear. Este protocolo requer apenas 10 nanogramas de começar RNA total e gera bibliotecas altamente consistentes representando transcrições todo.

Leia única e emparelhados End mRNA-Seq Bibliotecas Illumina de 10 nanogramas RNA total

Caracterizar a comunidade microbiana tem sido um objetivo de longa data na microbiologia ambiental. Os métodos de sequenciamento de última geração agora permitem a caracterização de comunidades microbianas em uma profundidade sem precedentes, com custo e mão de obra mínimos. Detalhamos aqui nossa abordagem para sequenciar genes de RNA ribossômico 16S bacteriano usando um sequenciador de bancada.

O sequenciamento de próxima geração de 16S RNA ribossomal Gene amplicons

Este protocolo descreve um método para mapeamento pre-mRNA' a extremidade 3 sites de processamento.

3' final de preparação da biblioteca com A-seq2 de sequenciamento

Sequenciamento proviral individual completo (aletas) fornece um método eficiente e de alta produtividade para a amplificação e sequenciamento de single, perto de provírus completos de HIV-1 (intactos e com defeito) e permite a determinação do seu potencial replicação-competência. FLIPS supera as limitações dos ensaios anteriores projetados para sequenciar o reservatório latente do HIV-1.

Amplificação de perto completos provírus HIV-1 para a próxima geração de sequenciamento

Long-leitura sequências facilitam grandemente a montagem de genomas complexas e caracterização da variação estrutural. Nós descrevemos um método para gerar as sequências ultra-longa pelas plataformas de sequenciamento baseado em nanopore. A abordagem adota uma extração de DNA otimizada seguida de preparações de biblioteca modificada para gerar centenas de mil leituras com cobertura moderada de células humanas.

Sequenciamento de leitura ultralonga para análise de DNA genômico completo

O sequenciamento de nanopore é uma nova tecnologia que permite o sequenciamento econômico em locais remotos e configurações pobres em recursos. Aqui, nós apresentamos um protocolo para arranjar em seqüência dos mRNAs do sangue inteiro que é compatível com tais circunstâncias.

Sequenciamento de mRNA de sangue total usando sequenciamento de nanoporos

Este protocolo descreve um fluxo de trabalho de culturas de células ex vivo ou in vitro para pré-processamento de dados transcriptômicos para triagem de drogas baseada em transcriptoma custo-efetivo.

Triagem Transcriptomic-Based Drug Screening Custo-Eficiente

O estudo atual descreve um fluxo de trabalho para sequenciamento nanoporoso de plasmídeos quando a sequência cromossômica já é conhecida ou não tem interesse. Uma etapa inicial de enriquecimento de DNA plasmídico garante a profundidade ótima da sequência plasmídica e maximiza o número de amostras que podem ser sequenciadas por célula de fluxo.

Sequenciamento e montagem de plasmídeos de leitura longa usando fluxos de trabalho baseados em sequenciamento nanopore

O presente estudo descreve um método simples de detectar níveis endógenos de fosforilação de Rab10 pela rica em leucina repetição quinase 2.

Deteção de fosforilação Rab10 pela atividade LRRK2 utilizando SDS-PAGE com um Tag de ligação de fosfato

Dentre as mais adversas condições de stress que organismos encontram durante seu ciclo de vida envolve o acúmulo de oxidantes. Durante o estresse oxidativo, células dependem fortemente de chaperones moleculares. Aqui, apresentamos os métodos usados para investigar redox-regulado antiatividade agregação, bem como para acompanhar mudanças estruturais que regulam a função de acompanhante usando HDX-MS.

Definição da atividade de chaperona regulada por redox de Hsp33 e mapeamento de alterações conformacionais em Hsp33 usando espectrometria de massa de troca de hidrogênio-deutério

Aprenda sequenciamento de amplicon com tecnologias de leitura longa para diagnóstico de tuberculose resistente a medicamentos. Este método oferece resultados precisos em ambientes com recursos limitados.

Sequenciamento de Amplicon usando as tecnologias de sequenciamento de leitura longa

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