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 ...

The overall goal of this method is to amplify 16 SRRNA from human milk and other low biomass samples using a semi-automated protocol. This method can help answer key questions in the development and perturbations of the human microbiome as it relates both to health and disease. The main advantage of the technique is that it's semi-automated, relatively easy to perform, and adaptable to multiple sample types including secretion, swabs, and stool.After thawing and alloquating whole milk according to the text protocol, spin the samples at 5000 times G and four degrees Celsius for 10 minutes to pellet the cells. Use a plastic spatula or large bore pipette tip to remove the top fat layer. Then without disturbing the pellet, remove all the supernatant, except for 100 microliters.With one milliliter of PBS, re-suspend and wash the pellet. Then prepare a negative control by adding one milliliter of sterile PBS to a five milliliter tube. Transfer the suspension to a clean one point five milliliter centrifuge tube and spin it at 5000 times G at room temperature for one minute.Then use a 1000 microliter sterile filtered pipette tip to discard the entire supernatant fatty layer. To disrupt and homogenize the sample, add 600 microliters of lysis buffer containing beta mercaptoethanol to the pellet and transfer the suspension to a bead tube. Prepare one positive control with lysis buffer and 20 microliters of the bacterial mock sample.Vortex all the bead tubes vigorously for 15 seconds. Then heat the samples on a temperature controlled vortexer at 37 degrees Celsius and 700 to 800 RPM for 10 minutes. Load the tubes into the automated sample disrupter adaptor set.Then bead beat the samples at 30 hertz for one minute. Disassemble the adaptor set and manually switch it with the sample plates from the left robotic arm to the right robotic arm of the instrument. Bead beat the tubes at 30 hertz for another minute then centrifuge the tubes at 17, 200 times G and 25 degrees Celsius for three minutes.Using a 200 microliter pipette, remove all of the supernatant, taking care not to take any of the fatty layer at the top or the bead residue at the bottom of the sample. Apply the supernatant to homogenizer columns. Then centrifuge the columns at maximum speed and 25 degrees Celsius for three minutes.Transfer 350 microliters of eluid to a two milliliter manufacturers microcentrifuge tube. For isolating DNA according to the text protocol, cut off the lids and smooth the edges of the individual spin columns for DNA and RNA. Insert the DNA spin column without the collection tube into the rotor adapter.Then discard the collection tube. Next, insert the vortexed two milliliter tubes into a shaker, following the automated DNA/RNA purification instrument's loading chart per the manufacturer's instructions. Label one point five milliliter collection tubes and insert them into rotor adapters.Then set the rotor adapters into the centrifuge following the automated DNA/RNA purification instrument's loading chart. After inserting filter tips, RNase free water, and the reagents into the instrument's reagent block, close the instrument's lid and run the custom DNA purification program on the instrument. When the reaction is complete, collect the sample containing tubes from the shaker, and if any sample is left, store it at minus 20 degrees Celsius for short term storage or minus 80 degrees Celsius for long term storage.After preparing the work area, and the PCR master mix, according to the text protocol, load 50 to 100 microliters of DNA samples into a 32-well instrument sample adapter, following the 96-well plate map. For each PCR plate, set up two negative controls by pipetting 30 microliters of PCR water in a clean one point five milliliter collection tube. Then place all the samples on the 32-well instrument sample adapter with caps locked in the open position.On the instrument's reagent adapter, remove the cap of each reverse primer with specific bar code numbers one at at time. To load the robotic liquid handler, place the reagent adapter in position B1.In order to avoid an edge effect, carefully place one edge of the adapter against the grip side and slowly bring the other edge down. Make sure to push on all the corners of the adapters.Place the sample adapter in position C1, making sure to push on all the corners of the adapters. Then vortex the five milliliter master mix. Open the cap and place it in position A on the instrument's master mix and reagent block.Place the PCR plate on the 96-well instrument's adapter that is intended to hold half skirted PCR plates. Then start the run and save it as a new file. When the run is complete, verify that there were no errors.Then use 12 or eight strip caps to seal the plate. Vortex vigorously and use a 96-well plate spinner to spin down the plates for one minute. Finally, place the plate on ice until it is moved to the post amplification room and loaded onto the thermal cycler.Following PCR amplification, carry out quality control and library preparation according to the text protocol. The protocol's first QC step follows PCR amplification of the 16SV4 region. As shown here, one microliter of PCR product from each sample, was analyzed by electrophoresis to confirm that it was within the expected size range of 350 to 450 base pairs.Some human milk samples generated lower amounts of specific product, suggesting either low levels of extracted microbial DNA in those samples or carryover of PCR inhibitors during extraction. Quantitation of specific product for each sample was essential for determining its required volume for equal molar pooling of samples for sequencing. A pooled library for targeted sequencing is usually dominated by a specific PCR product.If there is a significant amount of nonspecific product in the library, a gel purification step should be added to the workflow. Sequencing results demonstrate high diversity in the taxa associated with the human milk microbiome and variability in the number of sequencing reed counts for each sample. In contrast, the taxa composition and reed counts for the bacterial mock, were comparable to results obtained for the mock in previous workflow runs, suggesting that the observed variability for the human milk samples is an authentic experimental result and not a function of intrinsic workflow variability.If leaving a sequencer running overnight this technique can be done in two days, for 12 to 24 samples, if all samples amplify into the initial PCR run. If not, another shell day is needed. When attempting this procedure, it is important to travel only from the pre-PCR area to the post-PCR area to avoid contamination.It is also critical to change gloves frequently to avoid any cross contamination. Don't forget that working with human samples can be hazardous and precautions, such as proper protective equipment and bio safety cabinets, should always be used.

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