This work was funded by the USDA-ARS (CRIS 2030-21430-008-00D). TS is supported by the NSF Graduate Research Fellowship Program.

1. Collection and Separation of Root Endosphere, Rhizosphere, and Soil Samples
<ol>
	<li>Prior to entering the field, autoclave ultrapure water (at least 90 mL of water per sample) to sterilize. Prepare epiphyte removal buffer (at least 25 mL per sample) by adding 6.75 g of KH2PO4, 8.75 g of K2HPO4, and 1 mL of Triton X-100, to 1 L of sterile water. Sterilize the buffer using a vacuum filter with 0.2 &#181;m pore size.
	<ol>
		<li>For steps 1.2 to 1.5, wear clean gloves sterilized with ethanol at all times and replace the gloves between each sample to prevent contamination. Sterilize all equipment with 70% ethanol and wipe clean all equipment between samples. Before sampling, determine the optimal sampling depth for your experiment, and be consistent with all soil and root collections.</li>
	</ol>
	</li>
	<li>To collect bulk soil samples, use an ethanol-sterilized soil core collector to obtain soil that is free of plant roots by collecting a core approximately 23 to 30 cm from the base of the plant.</li>
	<li>Transfer the soil to a plastic bag, homogenize the soil by gentle shaking, and transfer an aliquot of the soil sample (approximately 600 mg) to fill one 2 mL tube. Immediately place the 2 mL tube on dry ice or flash freeze the tube in liquid N2 until ready to proceed with DNA extraction (step 2).
	<ol>
		<li>In some environments, the surrounding soil can contain plant material. In this case, use a sterilized 2 mm sieve to separate the plant debris from the soil prior to placing it in the plastic bag.</li>
	</ol>
	</li>
	<li>To collect the root and rhizosphere, use an ethanol-sterilized shovel to dig up the plant, taking care to obtain as much of the root as possible. Depth is dependent upon the plant. While small plants such as wheat can be removed by digging several centimeters, larger plants such as sorghum may require 30 cm or more. Gently shake off excess soil from the roots until there is approximately 2 mm of soil adhering to the root surface. 
	NOTE: Take care when working with small plants, with fragile roots, or in dry, high-clay content soils. Ideally, there should only be a thin layer of soil remaining on the roots after shaking. If large aggregates of soil remain, a rubber mallet can be used to dislodge the soil by gently hitting the base of the shoot. If the amount of soil remaining after this process exceeds or falls short of 2 mm, the approximate thickness should be noted.</li>
	<li>For large plants, use sterile scissors and/or shears to cut a representative subsection of roots and place a minimum of 500 mg of root tissue into a 50 mL conical vial. For smaller grasses, place the entire root system into the vial. Add enough epiphyte removal buffer to cover the roots, then immediately place the sample on dry ice or flash freeze the sample in liquid N2. 
	NOTE: Take care not to overfill the 50 mL conical vial, as it will make washing step difficult. There should be enough empty space such that the epiphyte buffer is able to flow to the bottom, surround the roots throughout the vial, and cover the top. Because some grasses have more root biomass than will fit into a 50 mL conical vial, a subsection of the roots should be collected. However, it should be noted that cutting the roots could lead to endophytic bacteria being washed out into the rhizosphere fraction, so breaking roots should be minimized. If samples are not processed immediately after returning to lab, they can be stored at -80 &#176;C.</li>
	<li>To separate the rhizosphere from the roots, thaw the root sample on ice, then sonicate the root samples at 4 &#176;C for 10 min with pulses of 160 W for 30 s, separated by 30 s. Transfer the roots into a chilled (4 &#176;C), clean 50 mL tube using sterile forceps. Do not dispose the original tube with buffer and soil, which is the rhizosphere fraction (Figure 1).</li>
	<li>Centrifuge the tube containing buffer and rhizosphere for 10 min at 4 &#176;C, 4,000 x g. Decant the supernatant, flash freeze the tube containing the rhizosphere fraction in liquid N2, and store the rhizosphere fraction at -80 &#176;C until ready to proceed with DNA extraction (step 2).</li>
	<li>To wash the roots, add approximately 20 mL of chilled (4 &#176;C) sterile water to the root fraction. Wash the root by shaking vigorously (by hand or mixer, for 15 - 30 s), and then drain the water.</li>
	<li>Repeat this step at least twice, until no soil remains on the root surface. If the DNA extraction (step 2) is not performed immediately, wrap the roots in sterile aluminum foil, flash freeze the roots in liquid N2, and store the samples at -80 &#176;C until ready to proceed with DNA extraction.</li>
</ol>
2. DNA Extraction
NOTE: Throughout steps 2 and 3, clean gloves sterilized with ethanol should be worn at all times and all work should be performed on a surface sterilized with ethanol.
<ol>
	<li>Extract DNA from the soil and rhizosphere samples.
	<ol>
		<li>Use a sterile spatula to quickly transfer 250 mg of soil and rhizosphere from steps 1.3 and 1.7 into separate collection tubes provided in a commercial DNA isolation kit designed for extraction from soil, then proceed with DNA isolation using the kit supplier&#39;s protocol.</li>
		<li>After eluting the DNA in the elution buffer supplied by the DNA isolation kit, store the DNA at -20 &#176;C until ready to proceed with step 3.</li>
	</ol>
	</li>
	<li>Extract DNA from the root samples.
	<ol>
		<li>Chill a sterilized mortar and pestle using liquid N2. Measure out 600 to 700 mg of root tissue and place the tissue into the mortar. Carefully, add enough liquid N2 to cover the roots.</li>
		<li>Grind the roots into small pieces. Continue the process of adding liquid N2 and grinding (at least two times, be consistent between samples), until the roots are a fine powder. Ensure that the root tissue does not thaw during this step. 
		Caution: Use appropriate personal protective equipment (lab coat, protective eyewear, and cryogenic gloves) when working with liquid N2. 
		NOTE: For a low-quality DNA extraction, it can be beneficial to grind excess roots into powder and store the powder at -80 &#176;C.</li>
		<li>Quickly, before the root powder begins to thaw, use a sterile spatula to transfer the root powder into pre-weighed 1.5 mL tubes on ice. Record the weight of the tube and powder. Typically, 300 - 400 mg of powder is transferred.</li>
		<li>Use a sterile spatula to quickly transfer 150 mg of root powder to the collection tube provided in a commercial DNA isolation kit designed for extraction from soil, then proceed with DNA isolation using the kit supplier&#39;s protocol. 
		NOTE: For some root samples, there can be a high concentration of organics remaining in the DNA pellet, which prevents the amplification of the DNA during PCR, especially when a different DNA extraction protocol (e.g., CTAB extraction) is used. If necessary, clean the DNA by following the instructions provided in the environmental DNA clean-up kit.</li>
	</ol>
	</li>
	<li>Measure the concentration of all DNA samples using a high-sensitivity benchtop fluorometer.
	<ol>
		<li>Add 1 - 20 &#181;L of each eluted DNA sample into tubes provided in the dsDNA high-sensitivity assay kit. Add fluorometer working solution (1:200 dye:buffer) up to 200 &#181;L.</li>
		<li>Prepare two additional tubes containing 10 &#181;L of DNA standard 1 (0 ng/&#181;L DNA) or 10 &#181;L of standard 2 (100 ng/&#181;L), and add 190 &#181;L of fluorometer working solution to each standard.</li>
		<li>Measure the concentration of the standards and each sample. If it is not done automatically, calculate the DNA concentration from the absorbance output by a linear regression of the two standards.</li>
	</ol>
	</li>
</ol>
3. Amplicon Library Preparation and Submission
<ol>
	<li>Set up materials for the amplification reaction.
	<ol>
		<li>Thaw DNA samples at 4 &#176;C and keep them on ice throughout step 3. Randomize the order of DNA samples to minimize bias due to the location on the PCR plate (Table 2.)</li>
		<li>In a 96-well PCR plate, dilute DNA from each sample in molecular-grade water to 5 ng/&#181;L in a total volume of 20 &#181;L. Add 20 &#181;L of molecular-grade water to the four corner wells as negative controls for amplification (blanks) (Table 2).</li>
		<li>Arrange the barcoded primers (10 &#181;M) in either PCR strip tubes or a 96-well plate such that they can be added with a multi-channel pipette (Figure 2).</li>
		<li>Prepare sufficient PCR master mix to amplify each DNA sample in triplicate. Prepare 1.5 &#181;L of BSA (20 mg/mL), 37.5 &#181;L of pre-made 2x master mix (composed of PCR buffer, MgCl2, dNTPs, and&#160;Taq&#160;DNA polymerase), 0.57 &#181;L of chloroplast PNA (100 &#181;M), 0.57 &#181;L of mitochondrial PNA (100 &#181;M), and 25.86 &#181;L of molecular grade water.</li>
		<li>Pour the master mix into a sterile 25 mL of multichannel pipette reservoir and distribute 66 &#181;L of master mix into each well of a new 96-well PCR plate using a multichannel pipette. 
		NOTE: When calculating reagent volumes for the master mix, make sure to also include the 4 blank wells per plate.</li>
		<li>Using a multi-channel pipette, add 6 &#181;L of 5 ng/&#181;L DNA (from the normalized DNA plate) to the master mix plate. Then add to the master plate 1.5 &#181;L of 10 &#181;M forward primer such that each column has a different forward barcode, and 1.5 &#181;L of 10 &#181;M reverse primer such that each row has a different reverse barcode (Figure 2). 
		NOTE: Prior to adding primers, the randomized plates and master mix could be used to amplify the ITS or ITS2 fungal genes if different primers were added. If this is the case, a similar primer design can be used.</li>
		<li>Spin down the plate briefly at 3,000 x g. Use a multi-channel pipette to mix gently, then divide into three plates with 25 &#181;L of reaction mixture. 
		NOTE: Although three replicates are not strictly necessary, it decreases the impact of technical variability.</li>
	</ol>
	</li>
	<li>Amplify the DNA in each plate using a thermocycler set to the following conditions: 180 s at 98 &#176;C, 30 cycles of: 98 &#176;C for 45 s (denaturing), 78 &#176;C for 10 s (PNA annealing), 55 &#176;C for 60 s (primer annealing), and 72 &#176;C for 90 s (extension), then 600 s at 72 &#176;C followed by a 4 &#176;C hold step. After the amplification, pool the three replicate plates into one single 96-well plate.</li>
	<li>Quantify the DNA using high-sensitivity fluorometer reagents in a 96-well plate reader.
	<ol>
		<li>Add 2 &#181;L of each PCR product to a 96-well microplate, along with 98 &#181;L of fluorometer working solution (1:200 dye:buffer). Include 4 wells as standards: 5 &#181;L of DNA standard 1 (0 ng/&#181;L DNA), 1 &#181;L of standard 2 (10 ng/&#181;L), 2 &#181;L of standard 2 (20 ng/&#181;L), and 5 &#181;L of standard 2 (50 ng/&#181;L). Then add fluorometer working solution for a final volume of 100 &#181;L. 
		NOTE: Each sample can be measured using a benchtop fluorometer as described in step 2.3 if a plate reader is not available.</li>
		<li>Calculate the DNA concentration from the absorbance output by a linear regression of the four standards.</li>
		<li>For the successfully amplified barcoded products (those that have a concentration greater than 15 ng/&#181;L), pool 100 ng of each sample into a single 1.5 mL tube (Table 2).</li>
		<li>Calculate the average volume of samples added to the pool by using the =AVERAGE() function in a spreadsheet program. Add the volume of the &#34;blank&#34; PCR products to the pooled samples. 
		NOTE: Since the &#34;blank&#34; PCR products have their own unique barcode combinations, they can be sequenced to check for any laboratory contaminants.</li>
	</ol>
	</li>
	<li>Measure the concentration of the pooled product using a benchtop fluorometer as described in step 2.3, and take 600 ng of DNA and dilute in molecular-grade water to a final volume of 100 &#181;L in a 1.5 mL tube. Store the remaining pooled product at -20 &#176;C.</li>
	<li>Wash the 600 ng DNA aliquot by following the established PCR purification process with paramagnetic purification beads in a 96-well format with a few exceptions.
	<ol>
		<li>Make a fresh 600 &#181;L aliquot of 70% ethanol. Shake the bottle of magnetic beads to re-suspend beads that settle to the bottom.</li>
		<li>Add 1x volume (100 &#181;L) of bead solution to the 600 ng aliquot of DNA. Mix thoroughly by pipetting 10 times. Incubate for 5 min at room temperature.</li>
		<li>Place the tube onto the magnetic stand for 2 min (or until solution is clear) to separate beads from solution. While the tube is still in the magnetic stand, aspirate the clear supernatant carefully without touching the magnetic beads, and discard the clear supernatant. 
		NOTE: At this point, the amplicon products are bound to the magnetic beads. Any beads that are disturbed or lost during aspiration will result in a loss of DNA.</li>
		<li>Leave the tube in the magnetic stand and add 300 &#181;L of 70% ethanol to the tube; incubate at room temperature for 30 s. Aspirate out the ethanol and discard. Repeat this process, and remove all ethanol after the second wash. Remove the tube from the magnetic stand, and air dry for 5 min.</li>
		<li>Add 30 &#181;L of molecular-grade water to the dried beads and mix by pipetting 10 times. Incubate at room temperature for 2 min. Return the tube to the magnetic stand for 1 min to separate the beads from solution. Transfer the eluate to a new tube. 
		NOTE: Magnetic beads will not affect downstream reactions.</li>
	</ol>
	</li>
	<li>Measure the final concentration of cleaned, pooled DNA using a benchtop fluorometer as described in step 2.3. Dilute an aliquot to 10 nM in a final volume of 30 &#181;L, or to the concentration and volume preferred by the sequencing facility.</li>
	<li>Utilize the services of a sequencing facility to sequence the DNA on a NGS platform, 2 x 300 bp paired-end sequencing.</li>
</ol>

The authors have nothing to disclose.

This protocol demonstrates an established pipeline for exploring root endosphere, rhizosphere, and soil microbial community compositions, from field sampling to sample processing and downstream sequencing. Studying root-associated microbiomes presents unique challenges, due in part to the inherent difficulties in sampling from soil. Soils are highly variable in terms of physical and chemical properties, and different soil conditions can be separated by as little as a few millimeters28,<...

Plant-associated microbiomes consist of dynamic and complex microbial communities comprised of bacteria, archaea, viruses, fungi, and other eukaryotic microorganisms. In addition to their well-studied role in causing plant disease, plant-associated microbes can also positively influence plant health by improving tolerance to biotic and abiotic stresses, promoting nutrient availability, and enhancing plant growth through the production of phytohormones. For this reason, particular interest exists in characterizing the taxa that associate with plant root endospheres, rhizospheres, and the surrounding soil. While some microbes can be cultured in isolation on laboratory generated media, many cannot, in part because they may rely on symbiotic relationships with other microbes, grow very slowly, or require conditions that cannot be replicated in a lab environment. Because it circumvents the need for cultivation and is relatively inexpensive and high-throughput, sequence-based phylogenetic profiling of environmental and host-associated microbial samples has become a preferred method for assaying microbial community composition.
The selection of appropriate sequencing technologies provided by various next generation sequencing (NGS) platforms1 is dependent on the users&#39; needs, with important factors including: desired coverage, amplicon length, expected community diversity, as well as sequencing error-rate, read-length, and the cost-per-run/megabase. Another variable that needs to be considered in amplicon-based sequencing experiments is what gene will be amplified and what primers will be used. When designing or choosing primers, researchers are often forced to make tradeoffs between the universality of amplification and the taxonomic resolution achievable from the resulting amplicons. For this reason, these types of studies often chose primers and markers that selectively target specific subsets of the microbiome. Evaluating the composition of bacterial communities is commonly accomplished by sequencing one or more of the hypervariable regions of the bacterial 16S rRNA gene2,3. In this study, we describe an amplicon based sequencing protocol developed for a NGS platform that targets the 500 bp V3-V4 region of the bacterial 16S rRNA gene, which allows for broad amplification of bacterial taxa while also providing sufficient variability to distinguish between different taxa. Additionally, this protocol can easily be adapted for the use with other primer sets, such as those targeting the ITS2 marker of fungi or the 18S rRNA subunit of eukaryotes.
While other approaches such as shotgun metagenomics, metatranscriptomics, and single-cell sequencing, offer other advantages including resolved microbial genomes and more direct measurement of community function, these techniques are typically more expensive and computationally intensive than the phylogenetic profiling described here4. Additionally, performing shotgun metagenomics and metatranscriptomics on root samples yields a large percentage of reads belonging to the host plant genome, and methods to overcome this limitation are still being developed5,6.
As with any experimental platform, amplicon-based profiling can introduce a number of potential biases which should be considered during the experimental design and data analysis. These include the methods of sample collection, DNA extraction, selection of PCR primers, and how library preparation is performed. Different methods can significantly impact the amount of usable data generated, and can also hinder the efforts to compare results between studies. For example, the method of removing rhizosphere bacteria7 and the use of different extraction techniques or choice of DNA extraction kits8,9 have been shown to significantly impact downstream analysis, which leads to different conclusions regarding which microbes are present and their relative abundances. Since amplicon-based profiling can be customized, making comparisons across studies can be challenging. The Earth Microbiome Project has suggested that researchers studying complex systems such as the plant-associated microbiome would benefit from the development of standardized protocols as a means of minimizing the variability caused by the application of different methods between studies10,11. Here, we discuss many of the above topics and offer suggestions as to best practices where appropriate.
The protocol demonstrates the process of collecting soil, rhizosphere, and root samples from Sorghum bicolor and extracting DNA using a well-established DNA isolation kit11. Additionally, our protocol includes a detailed amplicon sequencing workflow, using a commonly utilized NGS platform, to determine the structure of the bacterial communities12,13,14. This protocol has been validated for the use in a wide range of plant hosts in a recent published study of the roots, rhizosphere, and associated-soils of 18 monocot species including Sorghum bicolor, Zea mays, and Triticum aestivum15. This method has also been validated for use with other marker genes, as demonstrated by its successful application to studying the fungal ITS2 marker gene in studies of the agave microbiome16,17 and strawberry microbiome18.

<table><tbody><tr><td>0.1-10/20 &amp;micro;L filtered micropipette tips</td><td>USA Scientific</td><td>1120-3810</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5510</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>10 &amp;micro;L multi-channel pipette</td><td>Eppendorf</td><td>3122000027</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>10 &amp;micro;L, 100 &amp;micro;L, and 1000 &amp;micro;L micropipettes</td><td>Eppendorf</td><td>3120000909</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>100 &amp;micro;L multi-channel pipette</td><td>Eppendorf</td><td>3122000043</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>1000 &amp;micro;L filtered micropipette tips</td><td>USA Scientific</td><td>1122-1830</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>2 mL microcentrifuge tubes</td><td>USA Scientific</td><td>1620-2700</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>2 mm soil sieve</td><td>Forestry Suppliers</td><td>60141009</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>200 &amp;micro;L filtered micropipette tips</td><td>USA Scientific</td><td>1120-8810</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>25 mL reservoirs</td><td>VWR International LLC</td><td>89094-664</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>50 mL conical vials</td><td>Thermo Fisher Scientific</td><td>352098</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>500 mL vacuum filters (0.2 &amp;micro;m pore size)</td><td>VWR International LLC</td><td>156-4020</td><td></td></tr><tr><td>96-well microplates</td><td>USA Scientific</td><td>655900</td><td></td></tr><tr><td>96-well PCR plates</td><td>BioRad</td><td>HSP9631</td><td></td></tr><tr><td>Agencourt AMPure XP beads</td><td>Thermo Fisher Scientific</td><td>NC9933872</td><td>Instructions for use:&lt;br/&gt; https://www.beckmancoulter.com/wsrportal/ajax/downloadDocument/B37419AA.pdf?autonomyId=TP_DOC_150180&lt;br/&gt;&amp;amp;documentName=B37419AA.pdf</td></tr><tr><td>Aluminum foil</td><td>Boardwalk</td><td>7124</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Analytical scale with 0.001 g resolution</td><td>Ohaus Pioneer</td><td>PA323</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Bioruptor Plus ultrasonicator</td><td>Diagenode</td><td>B01020001</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) 20 mg/mL</td><td>New England Biolabs</td><td>B9000S</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5811000908</td><td>Including 50mL and 96-well plate bucket adapters</td></tr><tr><td>Cryogenic gloves</td><td>Millipore Sigma</td><td>Z183490</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>DNeasy PowerClean kit (optional)</td><td>Qiagen Inc.</td><td>12877-50</td><td>Previously MoBio</td></tr><tr><td>DNeasy PowerSoil kit</td><td>Qiagen Inc.</td><td>12888-100</td><td>Previously MoBio</td></tr><tr><td>Dry ice</td><td>Any</td><td>NA</td><td></td></tr><tr><td>DynaMag-2 magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td>Do not substitute</td></tr><tr><td>Ethanol</td><td>VWR International LLC</td><td>89125-188</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Gallon size freezer bags</td><td>Ziploc</td><td>NA</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Gemini EM Microplate Reader</td><td>Molecular Devices</td><td>EM</td><td>Can use another fluorometer that reads 96-well plates from the top.</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P3786</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Lab coat</td><td>Workrite</td><td>J1367</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Liquid N&lt;sub&gt;2&lt;/sub&gt;</td><td>Any</td><td>NA</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Liquid N&lt;sub&gt;2&lt;/sub&gt; dewar</td><td>Thermo Fisher Scientific</td><td>4150-9000</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Milli-Q ultrapure water purification system</td><td>Millipore Sigma</td><td>SYNS0R0WW</td><td></td></tr><tr><td>Mini-centrifuge</td><td>Eppendorf</td><td>5404000014</td><td></td></tr><tr><td>Molecular grade water</td><td>Thermo Fisher Scientific</td><td>4387937</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Mortars</td><td>VWR International LLC</td><td>89038-150</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Nitrile gloves</td><td>Thermo Fisher Scientific</td><td>19167032B</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Paper towels</td><td>VWR International LLC</td><td>BWK6212</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>PCR plate sealing film</td><td>Thermo Fisher Scientific</td><td>NC9684493</td><td></td></tr><tr><td>PCR strip tubes</td><td>USA Scientific</td><td>1402-2700</td><td></td></tr><tr><td>Pestles</td><td>VWR International LLC</td><td>89038-166</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Plastic spatulas</td><td>LevGo, Inc.</td><td>17211</td><td></td></tr><tr><td>Platinum Hot Start PCR Master Mix (2x)</td><td>Thermo Fisher Scientific</td><td>13000014</td><td></td></tr><tr><td>PNAs - chloroplast and mitochondrial</td><td>PNA Bio</td><td>NA</td><td>Make sure to verify sequence bioinformatically</td></tr><tr><td>Protective eyewear</td><td>Millipore Sigma</td><td>Z759015</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Thermo Fisher Scientific</td><td>Q32854</td><td></td></tr><tr><td>Rubber mallet (optional)</td><td>Ace Hardware</td><td>2258622</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Shears or scissors</td><td>VWR International LLC</td><td>89259-936</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Shovel</td><td>Home Depot</td><td>2597400</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Soil core collector (small diameter: &amp;lt;1 inch)</td><td>Ben Meadows</td><td>221700</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Spray bottles</td><td>Santa Cruz Biotechnology</td><td>sc-395278</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Standard desalted barcoded primers (10 &amp;micro;M) (Table 1)</td><td>IDT</td><td>NA</td><td>4 nmole Ultramer DNA Oligo with standard desalting. NGS adapter and sequencing primer (Table 1) are designed for use with Illumina MiSeq using v3 chemistry.</td></tr><tr><td>Thermocycler</td><td>Thermo Fisher Scientific</td><td>E950040015</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>Can substitute with equivalent from other suppliers.</td></tr><tr><td>Weigh boats</td><td>Spectrum Chemicals</td><td>B6001W</td><td>Can substitute with equivalent from other suppliers.</td></tr></tbody></table>

exploring the root microbiome: extracting bacterial community data from the soil, rhizosphere, and root endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Performing the recommended protocol should result in a dataset of indexed paired-end reads that can be matched back to each sample and assigned to either a bacterial operational taxonomic units (OTU) or exact sequence variant (ESV, also referred to as amplicon sequence variant (ASV) and sub-operational taxonomic unit (sOTU)), depending on downstream analysis. In order to obtain high-quality sequence data, care must be taken at each step to maintain consistency between samples and minimize...

Watch this Scientific Journal Video about Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere at JoVE.com

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere and Root Endosphere | Genetics | Video

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

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26.1K Views.  University of California, Berkeley. Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

Video: Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Environment

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Immunology and Infection

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

Developing Synergistic Multispecies Biofilms from Rhizosphere Soil

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.

Author Spotlight: Developing Synthetic Microbial Communities for Generating Second-Generation Biofertilizers

A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...

Community DNA Extraction from Bacterial Colonies

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Luisa Ikner
Traditional methods of analysis for microbial communities within soils have usually involved either cultural assays utilizing dilution and plating methodology on selective and differential media or direct count assays. Direct counts offer information about the total number of bacteria present, but give no information about the number or diversity of populations present within the community. Plate counts allow enumeration of total cultural or selected cultural populations, and hence provide information on the different populations present. However, since less than 1% of soil bacteria are readily culturable, cultural information offers only a piece of the picture. The actual fraction of the community that can be cultured depends on the medium chosen for cultural counts. Any single medium will select for the populations that are best suited to that particular medium.
In recent years, the advantages of studying community DNA extracted from soil samples have become apparent. This nonculture-based approach is thought to be more representative of the actual community present than culture-based approaches. In addition to providing information about the types of populations present, this approach can also provide information about their genetic potential. As with any technique, there are limitations to the data that can be obtained with DNA extraction. Therefore, many researchers now use DNA extraction in conjunction with direct and cultural counts to maximize the data obtained from an environmental sample.

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Traditional methods of analysis ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

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Chapters in this video

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

Community DNA Extraction from Bacterial Colonies