Name: guided protocol for fecal microbial characterization by 16s rrna-amplicon sequencing
Uploaded: 2018-03-19T13:30:00
Description: Watch this Scientific Journal Video about Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing at JoVE.com

Dette arbejde blev støttet i en del af programmet KERNEN (tilskud nr. 41/11), Israel Science Foundation (grant No. 908/15), og europæiske Crohns og Colitis organisation (ECCO).

Etisk godkendelse for studiet af Sheba lokale forskning etiske komité og alle metoder blev udført i overensstemmelse med de relevante retningslinjer og forordninger. Protokollen modtog en patientsamtykke undtagelse fra den lokale etiske Review Board, siden den fecal materiale, der blev brugt var allerede indsendt til mikrobiologi kernen som en del af klinisk workup og uden identificerbare patientinformation end alder, køn og mikrobielle resultater. Skrevet, blev indhentet informeret samtykke fra raske frivillige og de...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+Clostridium+difficile+infection+with+fecal+microbiota+transplantation.">Treating Clostridium difficile infection with fecal microbiota transplantation.</a> Clinical Gastroenterology and Hepatology. 9, 1044-1049 (2011).</li><li>Khoruts, A., Dicksved, J., Jansson, J. K., Sadowsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+composition+of+the+human+fecal+microbiome+after+bacteriotherapy+for+recurrent+Clostridium+difficile-associated+diarrhea.">Changes in the composition of the human fecal microbiome after bacteriotherapy for recurrent Clostridium difficile-associated diarrhea.</a> Journal of Clinical Gastroenterology. 44, 354-360 (2010).</li><li>Nood, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duodenal+infusion+of+donor+feces+for+recurrent+Clostridium+difficile.">Duodenal infusion of donor feces for recurrent Clostridium difficile.</a> The New England Journal of Medicine. 368, 407-415 (2013).</li><li>Koplan, J. P., Benfari Ferraro, M. J., Fineberg, H. V., Rosenberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+stool+cultures.">Value of stool cultures.</a> The Lancet. 316, 413-416 (1980).</li><li>Platts-Mills, J. A., Liu, J., Houpt, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+diagnostics+for+infectious+diarrhea.">New concepts in diagnostics for infectious diarrhea.</a> Mucosal Immunology. 6, 876-885 (2013).</li><li>Bokulich, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality-filtering+vastly+improves+diversity+estimates+from+Illumina+amplicon+sequencing.">Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing.</a> Nature Methods. 10, 57-59 (2013).</li><li>Leek, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+the+widespread+and+critical+impact+of+batch+effects+in+high-throughput+data.">Tackling the widespread and critical impact of batch effects in high-throughput data.</a> Nature Reviews Genetics. 11, (2010).</li><li>Dubilier, N., McFall-Ngai, M., Zhao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Create+a+global+microbiome+effort.">Create a global microbiome effort.</a> Nature. 526, 631-634 (2015).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+patterns+of+16S+rRNA+diversity+at+a+depth+of+millions+of+sequences+per+sample.">Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample.</a> PNAS. 108, 4516-4522 (2011).</li><li>Flores, G. E., Henley, J. B., Fierer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+PCR+approach+to+accelerate+analyses+of+human-associated+microbial+communities.">A direct PCR approach to accelerate analyses of human-associated microbial communities.</a> PloS One. 7, (2012).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QIIME+allows+analysis+of+high-throughput+community+sequencing+data.">QIIME allows analysis of high-throughput community sequencing data.</a> Nature Methods. 7, 335-336 (2010).</li><li>Callahan, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DADA2:+High-resolution+sample+inference+from+Illumina+amplicon+data.">DADA2: High-resolution sample inference from Illumina amplicon data.</a> Nature Methods. 13, 581-583 (2016).</li><li>Lozupone, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniFrac:+A+new+phylogenetic+method+for+comparing+microbial+communities.">UniFrac: A new phylogenetic method for comparing microbial communities.</a> Applied and Environmental Microbiology. 71, 8228-8235 (2005).</li><li>Lozupone, C., Lladser, M. E., Knights, D., Stombaugh, J., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniFrac:+An+effective+distance+metric+for+microbial+community+comparison.">UniFrac: An effective distance metric for microbial community comparison.</a> The ISME Journal. 5, 169-172 (2011).</li><li>Srinivasan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+16S+rRNA+gene+for+identification+of+a+broad+range+of+clinically+relevant+bacterial+pathogens.">Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens.</a> PloS One. 10, e0117617 (2015).</li><li>Hiergeist, A., Gl&#228;sner, J., Reischl, U., Gessner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+intestinal+microbiota:+culture+versus+sequencing.">Analyses of intestinal microbiota: culture versus sequencing.</a> ILAR Journal. 56, 228-240 (2015).</li><li>Didelot, X., Bowden, R., Wilson, D. J., Peto, T. E. A., Crook, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+clinical+microbiology+with+bacterial+genome+sequencing.">Transforming clinical microbiology with bacterial genome sequencing.</a> Nature Reviews Genetics. 13, 601-612 (2012).</li><li>Salipante, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+16S+rRNA+next-generation+sequencing+of+polymicrobial+clinical+samples+for+diagnosis+of+complex+bacterial+infections.">Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.</a> PloS One. 8, e65226 (2013).</li><li>Caporaso, J. 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16S rRNA-amplikon og metagenomics shotgun sekventering har vundet popularitet i klinisk Mikrobiologi programmer21,22,23. Disse teknikker er fordelagtige i deres øget evne til at fange bestemmelse og ikke-bestemmelse taxa, leverer data om den relative forekomst af patogene inokulum, og deres evne til at identificere flere netop en polymicrobial smitsomme fingerprint24. Fremskridt inden for microbiome forskning har genereret enorme mængder...

Har gjort koncentreret indsats for bedre at forstå microbiome diversiteten og tætheden, som et andet aspekt af fanger forskel og ligheder mellem personer i sund og patologiske betingelser. Alder2,3, geografi4, livsstil5,6, og sygdom5 viste sig at være forbundet med sammensætningen af gut microbiome, men mange betingelser og befolkninger har endnu ikke fuldt karakteriseret. For nylig er blevet rapporteret, at microbiome kan ændres til terapeutisk anvendelse7,8,9. Derfor, yderligere indsigt i forholdet mellem forskellige fysiologiske forhold og den mikrobielle sammensætning er det første skridt mod en optimering af mulige fremtidige ændringer.
Metoderne traditionelle mikrobielle kultur begrænses af lave udbytter10,11, og er udtænkt som en binær tilstand, hvor en bakterie er enten til stede i tarmen eller ej. Høj overførselshastighed DNA-baserede sekventering har revolutioneret mikrobiel økologi, gør det muligt for erobringen af alle medlemmer af mikrobielle samfund. Dog læse rækkefølge, længde og kvalitet er fortsat betydelige hindringer for nøjagtig taksonomi tildeling12. Høj overførselshastighed baseret eksperimenter kan desuden lider batch effekter, hvor målinger påvirkes af ikke-biologiske eller ikke-videnskabelige variabler13. I de seneste år, er der etableret flere programmer til at studere den human microbiome, herunder projektets amerikansk Gut, USA (US) Human Microbiome Project og Det Forenede Kongerige (UK) MetaHIT projekt. Disse initiativer har genereret enorme mængder af data, der ikke er let sammenlignelige på grund af en mangel på sammenhæng i deres strategier. En række internationale projekter såsom International Human Microbiome Consortium, projektets International Human Microbiome standarder og National Institute of Standards and Technology (NIST) forsøgte at løse nogle af disse spørgsmål14 , og udviklet standarder for microbiome målinger, som skal sætte opnåelsen af pålidelige reproduktive resultater. Beskrevet her er en integreret protokol af flere bredt anvendte metoder15,16 for 16S rRNA høj overførselshastighed sekventering (16S-seq) startende fra fecal prøvetagning igennem data analyser. Protokollen beskriver en kolonne-fri PCR metode, oprindeligt udviklet til direkte udtræk af planten DNA16, for at aktivere den samtidige håndtering af store mængder af fækal prøver i en forholdsvis kort tid med høj kvalitet amplificeret DNA for målrettet sekventering af mikrobielle variable V4 regionen på en fælles platform, sekventering. Denne protokol har til formål at guide forskere interesseret i at indlede brugen af 16S rRNA-amplikon sekventering i en robust, reproduktive, nem at bruge, detaljeret måde, ved hjælp af vigtige kontrol. At have en guidet og detaljerede trin-for trin protokol kan minimere batch effekt og således vil give mere sammenlignelige sekventering resultater mellem labs.

<table>
	<tbody>
		<tr>
			<td>Primers</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extraction solution</td>
			<td>Sigma-Aldrich</td>
			<td>E7526</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilution solution</td>
			<td>Sigma-Aldrich</td>
			<td>D5688</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa HiFi HotStart ReadyMix PCR Kit</td>
			<td>KAPABIOSYSTEMS</td>
			<td>KK2601</td>
			<td>PCR Master mix</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Reagent kit</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td>dsDNA quantify reagent</td>
		</tr>
		<tr>
			<td>MinElute Gel extraction kit</td>
			<td>Qiagen</td>
			<td>28606</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Amresco</td>
			<td>0710-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure Water Dnase and Rnase Free</td>
			<td>Biological Industries</td>
			<td>01-866-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Molecular probes</td>
			<td>Q32854</td>
			<td>dsDNA detecting kit</td>
		</tr>
		<tr>
			<td>High Sensitivity D1000</td>
			<td>Agilent Technologies</td>
			<td>Screen Tape 5067-5582</td>
			<td>separation and analysis</td>
		</tr>
		<tr>
			<td>Screen Tape Assay</td>
			<td>Agilent Technologies</td>
			<td>Reagents 5067-5583</td>
			<td>for DNA libraries</td>
		</tr>
		<tr>
			<td>PhiX Control v3</td>
			<td>Illumina</td>
			<td>15017666</td>
			<td>control library</td>
		</tr>
		<tr>
			<td>MiSeq Reagent Kit v2 (500 cycle)</td>
			<td>Illumina</td>
			<td>MS-102-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>E406-10mL-TAM</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL collection tubes</td>
			<td>SARSTEDT</td>
			<td>72.695.400</td>
			<td>Safe Seal collection tubes</td>
		</tr>
		<tr>
			<td>Plastic stick swab in PP test tube</td>
			<td>STERILE INTERIOR</td>
			<td>23117</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Machine</td>
			<td>Applied Biosystems</td>
			<td>2720 Thermal Cycler</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequncing Machine</td>
			<td>Illumina</td>
			<td>Miseq</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR workstation</td>
			<td>Biosan</td>
			<td>UV-cleaner</td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vortexer</td>
			<td>Scientific Industries</td>
			<td>Vortex-Genie 2</td>
			<td></td>
		</tr>
	</tbody>
</table>

guided protocol for fecal microbial characterization by 16s rrna-amplicon sequencing

Den menneskelige intestinal microbiome spiller en central rolle beskytte celler mod skader, behandling af energi og næringsstoffer, og at fremme immunitet. Afvigelser fra hvad der betragtes som en sund mikrobiota sammensætning (dysbiosis) kan forringe vitale funktioner fører til patologiske betingelser. De seneste og igangværende forskningsindsats har været rettet mod karakterisering af tilknytninger mellem mikrobielle sammensætning og menneskers sundhed og sygdom.
Fremskridt i høj overførselshastighed sekventering teknologier aktiverer karakterisering af gut mikrobielle sammensætning. Disse metoder omfatter 16S rRNA-amplikon sekvensering og shotgun sekventering. 16S rRNA-amplikon sekventering bruges til at profilere taksonomisk sammensætning, mens shotgun sekventering giver yderligere oplysninger om gen forudsigelser og funktionelle anmærkning. En fordel i at anvende en målrettet sekventering metode af 16S rRNA gen variable region er dens betydeligt lavere omkostninger sammenlignet med shotgun sekvensering. Sekvens forskelle i 16S rRNA gen bruges som en mikrobiel fingeraftryk til at identificere og kvantificere forskellige taxa i en individuel prøve.
Store internationale bestræbelser har hyret standarder til 16S rRNA-amplikon sekvensering. Men, flere undersøgelser rapport en fælles kilde til variation forårsaget af batch effekt. For at minimere denne effekt, uniformerede protokoller for prøvetagning, skal forarbejdning og sekventering gennemføres. Denne protokol foreslår integration af bredt anvendte protokoller fra fecal prøvetagning til dataanalyse. Denne protokol indeholder en kolonne-fri, direkte-PCR tilgang, der muliggør samtidige håndtering og DNA-ekstraktion af store mængder fækal prøver, sammen med PCR-amplifikation af V4-regionen. Derudover protokollen beskriver analyse rørledningen og indeholder et script ved hjælp af den nyeste version af QIIME (QIIME 2 version 2017.7.0 og DADA2). Denne trinvise protokollen har til formål at guide dem interesseret i at indlede brugen af 16S rRNA-amplikon sekventering i en robust, reproduktive, nem at bruge, detaljeret måde.

En skematisk illustration af protokollen er vist i figur 1.
Fremadrettet har vi samlet afføringsprøver fra indlagte patienter med mistænkt infektiøs diarré. Disse prøver blev indsendt til klinisk Mikrobiologi Lab på Sheba Medical Center mellem februar og maj 2015, som var tidligere beskrevet1. Afføringsprøver blev udsat for konventionel mikrobiologiske kultur udført...

Watch this Scientific Journal Video about Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing at JoVE.com

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

Dette manuskript beskriver en detaljeret standardiseret protokol af høj overførselshastighed 16S rRNA-amplikon sekvensering. Protokollen introducerer en integreret, uniformerede, gennemførlige og billig protokol fra fecal prøvetagning gennem data analyser. Denne protokol giver mulighed for analyse af store mængder prøver med strenge standarder og flere kontrolelementer.

Guidede protokol for fækal mikrobielle karakterisering af 16S rRNA-amplikon sekventering

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