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

We would like to thank Helty Adisetiyo, PhD and Shangxin Yang, PhD for the development of the protocol.Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) was provided by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) under Award Numbers UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC) and UM1AI106716 (IMPAACT LC), with co-funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and the National Institute of Mental Health (NIMH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

For all protocol steps, proper personal protective equipment (PPE) must be worn, and stringent contamination prevention approaches need to be taken. Observe flow of work from pre-amplification work areas to post-amplification work areas to minimize contamination of samples. All supplies used are sterile, free of RNase, DNase, DNA, and pyrogen. All pipette tips are filtered. A flowchart of the protocol steps is provided (Figure 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>

Targeted next-generation sequencing of 16S rRNA is a widely used, rapid technique for microbiome characterization18. However, many factors, including batch effects, environmental contamination, sample cross-contamination, sensitivity, and reproducibility can adversely affect experimental results and confound their interpretation7,19,20. To best facilitate robust 16S analyses, microbiome workflows must inc...

The microbial communities that colonize humans are believed to be critically important to human health and disease influencing metabolism, immune development, susceptibility to disease, and responses to vaccination and drug therapy1,2. Efforts to understand the influence of the microbiota on human health currently emphasize the identification of microbes associated with defined anatomic compartments (i.e., skin, gut, oral, etc.), as well as localized sites within these compartments3,4. Underpinning these investigative efforts is the rapid emergence and increased accessibility of next-generation sequencing (NGS) technologies that provide a massively parallel platform for analysis of the microbial genetic content (microbiome) of a sample. For many physiological samples, the associated microbiome is both complex and abundant (i.e., stool), but, for some samples, the microbiome is represented by low microbial biomass (i.e., human milk, lower respiratory tract) where sensitivity, experimental artefacts, and possible contamination become major issues. The common challenges of microbiome studies and appropriate experimental design have been the subject of multiple review articles5,6,7,8.
Presented herein is a robust NGS experimental pipeline based on targeted sequencing of the rRNA 16S V4 region9 to characterize the microbiome of human milk. Microbiome analysis of human milk is complicated not only by an inherently low microbial biomass10, but additionally by high levels of human DNA background11,12,13,14 and potential carryover of PCR inhibitors15,16 in extracted nucleic acid. This protocol relies on commercially available extraction kits and semi-automated platforms that can help minimize variability across sample preparation batches. It incorporates a well-defined bacterial mock community that is processed alongside samples as a quality control to validate each step in the protocol and provide an independent metric of pipeline robustness. Although the protocol as described is specific to the human milk samples, it is readily adaptable to other sample types including stool, rectal, vaginal, skin, areolar, and oral swabs10,17, and can serve as a starting point for researchers who wish to perform microbiome analyses.

<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

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

The protocol presented here includes important quality control (QC) steps to ensure that the data generated meet benchmarks for protocol sensitivity, specificity, and contamination control. The protocol&#39;s first QC step follows PCR amplification of the 16S V4 region (Figure 2). One &#181;L of PCR product from each sample was analyzed by electrophoresis to confirm that it was within the expected size range of 315 - 450 bp (Figure 2</str...

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A Method for Targeted 16S Sequencing of Human Milk Samples

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

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

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