Name: Sequenziamento degli ampliconi con le tecnologie di sequenziamento a lettura lunga (Video) | JoVE Journal
Uploaded: 2025-08-29T13:40:00.000+00:00
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Description: Scopri il sequenziamento degli ampliconi con tecnologie a lettura lunga per la diagnosi della tubercolosi farmacoresistente. Questo metodo offre profili di resistenza accurati e tempi di analisi ridotti.

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<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>
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<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>
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<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>
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</ol>

<table><tbody><tr><td>15 mL & Tubi Lo-binding da 1,5 mL o 2 mL</td><td>Eppendorf</td><td>EP0030108051-250EA</td><td></td></tr><tr><td>Scala da 1kb</td><td>Thermo Fisher Scientific</td><td>SM0311</td><td></td></tr><tr><td>Tubi a tappo a vite da 2 mL</td><td>Specialità Scientifiche Inc</td><td>P2TUB056C-0001.5ST</td><td></td></tr><tr><td>Perle di vetro sterili da 2 mm</td><td>Sigma-Aldrich</td><td>Z273627-1EA</td><td></td></tr><tr><td>Sferato XP AMPure</td><td>Beckman Colter</td><td>A63881</td><td></td></tr><tr><td>Deeplex Myc-TB&nbsp;</td><td>GenoScreen</td><td>20090205</td><td></td></tr><tr><td>Camera di elettroforesi</td><td>Thermo Fisher Scientific</td><td>A25977</td><td></td></tr><tr><td>Alcol etilico, puro</td><td>Sigma-Aldrich</td><td>E7023-500ML</td><td></td></tr><tr><td>Omogeneizzatore FastPrep</td><td>MP Biomedici</td><td>116005500</td><td></td></tr><tr><td>Punte delle pipette a filtro (10 & micro; L)&nbsp;</td><td>Bio-Smart Scientific</td><td>FT-10-R</td><td></td></tr><tr><td>Punte per pipette a filtro (1000 & micro; L)&nbsp;</td><td>Bio-Smart Scientific</td><td>FT-1000-R</td><td></td></tr><tr><td>Punte delle pipette filtranti (20 & micro; L)&nbsp;</td><td>Bio-Smart Scientific</td><td>FT-20-R</td><td></td></tr><tr><td>Punte di pipette filtro (200 & micro; L)&nbsp;</td><td>Bio-Smart Scientific</td><td>FT-200-R</td><td></td></tr><tr><td>Cella di flusso &nbsp;</td><td>Oxford Nanopore Technologies</td><td>FLO-MIN114</td><td></td></tr><tr><td>Blocco riscaldamento</td><td>Eppendorf</td><td>5382000031</td><td></td></tr><tr><td>Matrice InstaGene</td><td>Bio-Rad</td><td>BBRD7326030</td><td></td></tr><tr><td>Rack magnetico&nbsp;</td><td>Thermo Fisher Scientific&nbsp;</td><td>12321D</td><td></td></tr><tr><td>Microcentrifuga</td><td>Eppendorf&nbsp;</td><td>5406000046</td><td></td></tr><tr><td>Tirapiedi</td><td>Oxford Nanopore Technologies</td><td>MK1B</td><td></td></tr><tr><td>Kit di codifica a barre nativa V14&nbsp;</td><td>Oxford Nanopore Technologies</td><td>SQK-NBD114.24</td><td></td></tr><tr><td>NEB Blunt/TA Ligase Master Mix</td><td>NEB</td><td>M0367</td><td></td></tr><tr><td>Modulo di Legatura Rapida NEBNext</td><td>NEB</td><td>E6056</td><td></td></tr><tr><td>NEBNext Ultra II Modulo di riparazione estrema/dA-tailing</td><td>NEB</td><td>E7546</td><td></td></tr><tr><td>Acqua priva di nucleasi (NFW)</td><td>Thermo Fisher Scientific</td><td>AM9937</td><td></td></tr><tr><td>Pipette (P10, P20, P200 e P1000)</td><td>Eppendorf</td><td>EP3123000918-1EA</td><td></td></tr><tr><td>Tubi per il saggio dei Qubit</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Kit di test HS per Qubit dsDNA</td><td>Thermo Fisher Scientific</td><td>Q33231</td><td></td></tr><tr><td>Fluorometro a qubit</td><td>Thermo Fisher Scientific</td><td>D33226</td><td></td></tr><tr><td>Ceppo di gel sicuro di DNA SYBR</td><td>Thermo Fisher Scientific</td><td>S33102</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Thermo Fisher Scientific</td><td>17852</td><td></td></tr><tr><td>Universal Hood III transilluminatore (Sistema Molecular Imager Gel Doc XR)</td><td>Bio-Rad</td><td>170-8170</td><td></td></tr><tr><td>Miscelatore a vortice</td><td>Lasec</td><td>WMBB0L0E0216</td><td></td></tr></tbody></table>

sequenziamento degli ampliconi con le tecnologie di sequenziamento a lettura lunga

Amplicon Sequencing using the Long-Read Sequencing Technologies

Sequenziamento degli ampliconi con le tecnologie di sequenziamento a lettura lunga

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

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Qui si descrive un metodo per la preparazione di entrambi i singoli letti e associato fine Illumina mRNA-Seq librerie sequenziamento per l'analisi di espressione genica basata su amplificazione lineare dell'RNA T7. Questo protocollo richiede solo 10 nanogrammi di RNA totale di partenza e genera librerie altamente coerente rappresentanza trascrizioni intero.

Leggi singolo e accoppiamento finale mRNA-Seq Biblioteche Illumina da 10 nanogrammi di RNA totale

Caratterizzare la comunità microbica è stato un obiettivo di lunga data nella microbiologia ambientale. I metodi di sequenziamento di nuova generazione consentono ora la caratterizzazione delle comunità microbiche a una profondità senza precedenti, con costi e manodopera minimi. Descriviamo qui il nostro approccio al sequenziamento dei geni dell'RNA ribosomiale batterico 16S utilizzando un sequenziatore da banco.

Sequenziamento di prossima generazione del 16S RNA ribosomiale Gene ampliconi

Questo protocollo descrive un metodo per la mappatura pre-mRNA 3' fine elaborazione siti.

3' fine sequenziamento preparazione libreria con A-seq2

Full-Length individuo proviral sequenziamento (Flip) fornisce un metodo efficiente e ad alta produttività per l'amplificazione e sequenziamento del singolo, vicino a full-length provirus di HIV-1 (intatto e difettoso) e permette per la determinazione del loro potenziale replica-competenza. Salti mortali supera i limiti delle precedenti analisi progettate per sequenziare il serbatoio latente di HIV-1.

Amplificazione del vicino full-length provirus di HIV-1 per il sequenziamento di nuova generazione

Lungo-ha letto sequenze notevolmente facilitano l'assemblaggio di complessi genomi e caratterizzazione di variazione strutturale. Descriviamo un metodo per generare sequenze ultra-lunghi di piattaforme nanopore di sequenziamento. L'approccio adotta un'estrazione di DNA ottimizzata seguita da preparazioni di libreria modificata per generare centinaia di kilobase letture con moderata copertura da cellule umane.

Sequenziamento di lettura ultra-lungo per analisi di DNA Genomic intero

Il sequenziamento di nanopore è una nuova tecnologia che consente un sequenziamento conveniente in posizioni remote e impostazioni di scarsa risorse. Qui, presentiamo un protocollo per il sequenziamento di mRNA da sangue intero che è compatibile con tali condizioni.

Sequenziamento dell'mRNA da sangue intero utilizzando il sequenziamento di NanoPore

Questo protocollo descrive un flusso di lavoro che va dalle colture cellulari ex vivo o in vitro alla pre-elaborazione dei dati trascrittomici per uno screening farmacologico basato sul trascrittoma economicamente vantaggioso.

Screening farmacologico basato sulla trascrittomica efficiente in termini di costi

Lo studio attuale descrive un flusso di lavoro per il sequenziamento nanoporo dei plasmidi quando la sequenza cromosomica è già nota o non ha alcun interesse. Un primo passaggio di arricchimento del DNA plasmidico garantisce una profondità ottimale della sequenza plasmidica e massimizza il numero di campioni che possono essere sequenziati per cella di flusso.

Sequenziamento e assemblaggio di plasmidi a lunga lettura utilizzando flussi di lavoro basati sul sequenziamento nanopore

Lo studio presente descrive un metodo semplice per rilevare i livelli endogeni di Rab10 fosforilazione dalla leucina-ricche ripetere chinasi 2.

Rilevamento di fosforilazione di Rab10 di LRRK2 attività mediante SDS-PAGE con un Tag di fosfato-binding

Uno della più difficili condizioni di stress che gli organismi si incontrano durante la loro vita comporta un accumulo di ossidanti. Durante lo sforzo ossidativo, le cellule si basano pesantemente su chaperoni molecolari. Qui, presentiamo i metodi utilizzati per indagare la redox-regolati anti-aggregazione attività, nonché monitorare i cambiamenti strutturali che disciplinano la funzione di chaperone utilizzando HDX-MS.

Definizione attività di Chaperone Redox-regolati di Hsp33 e mappatura cambiamenti conformazionali su Hsp33 usando la spettrometria totale di scambio di idrogeno-deuterio

Scopri il sequenziamento degli ampliconi con tecnologie a lettura lunga per la diagnosi della tubercolosi farmacoresistente. Questo metodo offre profili di resistenza accurati e tempi di analisi ridotti.

Sequenziamento degli ampliconi con le tecnologie di sequenziamento a lettura lunga

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