Name: a method for targeted 16s sequencing of human milk samples
Uploaded: 2018-03-23T12:30:00
Description: Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

Vi vil gerne takke Helty Adisetiyo, ph.d.- og Shangxin Yang, ph.d. for udviklingen af protokollen. Samlede støtte til den internationale maternel Pediatric Adolescent AIDS kliniske forsøg gruppe (IMPAACT) blev leveret fra National Institute for allergi og smitsomme sygdomme (NIAID) af National Institutes of Health (NIH) under Award numre UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC) og UM1AI106716 (IMPAACT LC), med samfinansiering fra Eunice Kennedy Shriver nationale Institut for børns sundhed og menneskelige udvikling (NICHD) og National Institute of Mental Health (NIMH). Indholdet er udelukkende ansvarlig for forfattere og repræsenterer ikke nødvendigvis de officielle synspunkter af NIH.

Alle trin, protokol, passende personlige værnemidler (PPE) skal bæres, og strenge forurening forebyggelse tilgange skal tages. Observere strøm af arbejde fra før forstærkning arbejdsområder til efter forstærkning arbejdsområder for at minimere forurening af prøver. Alle leverancer bruges er sterile, fri for RNase, DNase, DNA og pyrogen. Alle pipette tips er filtreret. Et rutediagram i protokollen trin er fastsat (figur 1).
1. sample Lysis
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<ol>
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Mock microbial communities Available from: <a target="_blank" href="https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1">https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1</a> (2017)</li></ol>

Målrettet næste generations sekventering af 16S rRNA er en udbredt, hurtig teknik til microbiome karakterisering18. Men mange faktorer, herunder batch effekter, miljøforurening, prøve krydskontaminering, følsomhed og reproducerbarhed negativt kan påvirke eksperimentelle resultater og forvirre deres fortolkning7,19 , 20. bedst lette robust 16S analyser, microbiome arbejdsprocesser skal indarbejde god...

De mikrobielle samfund, at kolonisere mennesker menes at være afgørende betydning for menneskers sundhed og sygdom påvirker stofskiftet, immun udvikling, modtagelighed over for sygdom, og svar til vaccination og stofmisbrug behandling1, 2. bestræbelser på at forstå mikrobiota indflydelse på menneskers sundhed i øjeblikket understrege identifikation af mikrober tilknyttet defineret anatomisk rum (dvs., hud, gut, oral, etc.), samt lokaliserede sites inden for disse rum3,4. Understøttelse af disse efterforskningsmæssige indsats er den hurtige fremkomst og øget tilgængelighed af næste generation sequencing (NGS) teknologier, der giver et massivt parallel platform til analyse af de mikrobielle genetiske indhold (microbiome) af en stikprøve. For mange fysiologiske prøver, den tilknyttede microbiome er både komplekse og rigelige (dvs., fæces), men for nogle prøver, microbiome er repræsenteret ved lav mikrobielle biomasse (dvs., modermælk, nedre luftveje) hvor følsomhed, eksperimentelle artefakter og eventuel forurening bliver store spørgsmål. De fælles udfordringer microbiome undersøgelser og relevante eksperimentelle design har været genstand for flere review artikler5,6,7,8.
Præsenteres heri er en robust NGS eksperimentelle pipeline baseret på målrettede sekventering af rRNA 16S V4 region9 at karakterisere microbiome af modermælk. Microbiome analyse af modermælk er kompliceret, ikke kun ved en ifølge sagens natur lav mikrobielle biomasse10, men derudover ved høj niveauer af menneskelig DNA baggrund11,12,13,14 , og potentielle fremførsel af PCR-hæmmere15,16 i uddraget nukleinsyre. Denne protokol er afhængig af kommercielt tilgængelige udvinding kits og semi-automatiske platforme, der kan hjælpe med at minimere variation på tværs af prøven forberedelse partier. Sig indlemmer en veldefineret bakteriel mock Fællesskabets, behandles sideløbende med prøver som en kvalitetskontrol til at validere hvert trin i protokollen og give en uafhængig måling af rørledningen robusthed. Selv om protokollen som beskrevet er specifikke for de menneskelige mælkeprøver, det er let at tilpasse til andre prøve typer herunder taburet, rektal, vaginal, hud, areolar, og mundtlige svaberprøver10,17, og kan tjene som udgangspunkt for forskere, der ønsker at udføre microbiome analyser.

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">AllPrep RNA/DNA Mini Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">80204</td>
			<td style="width:395px;">DNA/RNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Eliminase</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:153px;">435532</td>
			<td>RNase, DNase, DNA decontaminant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Thermo Mixer</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td>temperature-controlled vortexer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Buffer RLT plus</td>
			<td style="width:115px;">Qiagen</td>
			<td>1053393</td>
			<td>guanidinium thiocyanate lysis buffer/ Part of Allprep kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#223;-Mercaptoethanol&#160;</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td>63689-25ML-F</td>
			<td>&#223;-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">LME Beads</td>
			<td style="width:115px;">MP Biomedicals</td>
			<td>116914050</td>
			<td>bead tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAgen TissueLyzer</td>
			<td style="width:115px;">Qiagen</td>
			<td>85300</td>
			<td>automated sample disruptor adapter set</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAshredder column</td>
			<td style="width:115px;">Qiagen</td>
			<td>&#160;79654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAgen RB tube</td>
			<td style="width:115px;"></td>
			<td></td>
			<td>manufacturer&#39;s microcentrifuge tube in kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAcube and related plasticware</td>
			<td style="width:115px;">Qiagen</td>
			<td>9001292</td>
			<td>automated DNA/RNA purification instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">DNA exitus plus</td>
			<td style="width:115px;">Applichem</td>
			<td>A7089</td>
			<td>non-enzymatic decontamination solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EB Buffer</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">19086</td>
			<td>elution buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAgility and related plasticware</td>
			<td style="width:115px;">Qiagen</td>
			<td>9001532</td>
			<td>robotic liquid handler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR water</td>
			<td style="width:115px;">MO BIO</td>
			<td>17000-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5PRIME HotMasterMix</td>
			<td style="width:115px;">Quantabio</td>
			<td>2200400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Barcoded reverse primers</td>
			<td style="width:115px;">Eurofin</td>
			<td style="width:153px;">No Catalog #&#39;s</td>
			<td>designed and ordered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">&#160;96 well PCR plate</td>
			<td style="width:115px;">USA scientific</td>
			<td style="width:153px;">1402-9708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tapestation 2200 and related plasticware</td>
			<td style="width:115px;">Agilent</td>
			<td>G2964AA</td>
			<td>automated DNA/RNA fragment analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D1000 reagents for Tapestation&#160;</td>
			<td style="width:115px;">Agilent</td>
			<td>5067-5585</td>
			<td>Sample buffer and ladder are part of this kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OneStep PCR Inhibitor Removal Kit&#160;</td>
			<td style="width:115px;">Zymo Research</td>
			<td style="width:153px;">50444470</td>
			<td>PCR inhibitor removal is done per the manufacturer&#39;s instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAquick PCR Purification Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">28104</td>
			<td>DNA clean up kit: silica-membrane-based purification of PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td style="width:153px;">Q32854</td>
			<td>dimethylsulfoxide-based dilution buffer and dye are part of this kit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Qubit Fluorometer</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NanoDrop</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td style="width:153px;"></td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MiSeq 300 V2 kit</td>
			<td style="width:115px;">Illumina</td>
			<td>15033624/15033626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MiSeq&#160;&#160;&#160;</td>
			<td style="width:115px;">Illumina</td>
			<td style="width:153px;">No Catalog #&#39;s</td>
			<td>next generation sequencer</td>
		</tr>
	</tbody>
</table>

a method for targeted 16s sequencing of human milk samples

Undersøgelser af mikrobielle samfund er blevet udbredt med udviklingen af relativt billig, hurtig og høj overførselshastighed sekvensering. Men som med alle disse teknologier, reproducerbare resultater afhænger en laboratorium arbejdsproces, der omfatter passende forholdsregler og kontrol. Dette er især vigtigt med lav-biomasse prøver, hvor forurener bakteriel DNA kan genererer vildledende resultater. I denne artikel beskrives en semi-automatiseret arbejdsproces til at identificere mikrober fra menneskelige bryst mælkeprøver ved hjælp af målrettede sekventering af 16S ribosomale RNA (rRNA) V4 region på en lav og midten af throughput skala. Protokollen beskriver prøveforberedelse fra sødmælk herunder: prøve lysis, nukleinsyre udvinding, forstærkning af regionen V4 af 16S rRNA gen, og biblioteket forberedelse med foranstaltninger til kvalitetskontrol. Vigtigere, protokol og diskussion overveje spørgsmål, der er iøjnefaldende udarbejdelse og analyse af lav-biomasse prøver herunder passende positive og negative kontroller, PCR hæmmer fjernelse, prøve forurening af miljøet, reagens, eller eksperimentelle kilder, og eksperimenterende bedste praksis skal sikre reproducerbarhed. Mens protokollen som beskrevet er specifikt for humant mælkeprøver, er det kan tilpasses til mange lav - og høj-biomasse prøve typer, herunder prøver, der opsamlet på podninger, frosne pæn eller stabiliseret i en buffer, konservering.

Protokollen præsenteres her indeholder vigtige kvalitetskontrol (QC) trin for at sikre, at data genereret mødes benchmarks for protokollens følsomhed, specificitet og forurening kontrol. Protokollens første QC skridt følger PCR-amplifikation af regionen 16S V4 (figur 2). Én µL PCR produkt fra hver prøve blev analyseret af elektroforese at bekræfte at det var inden for det forventede størrelse 315-450 bp (figur 2, rød p...

Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

A Method for Targeted 16S Sequencing of Human Milk Samples

En semi-automatiseret arbejdsproces præsenteres for målrettede sekventering af 16S rRNA fra modermælk og andre lav-biomasse prøve typer.

En metode til målrettede 16S sekvensering af modermælk prøver

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

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Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

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The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

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There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

En hurtig metode til Blood Processing at øge udbyttet af Plasma-peptid niveauer i Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Swab Sampling metode til påvisning af menneskelige Norovirus på overflader

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

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Magnetisk omrører metode til påvisning af<em&gt; trikiner</em&gt; Larver i Muscle Prøver

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

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Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

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