This project was support in part by Johns Hopkins University, Office of the Provost through the Gateway Science Initiative.

Note: Metagenomic DNA is purified from soil (Baltimore County, Maryland) using a commercially available soil genomic isolation kit (see Table of Materials). Using an UV spectrophotometer (see Table of Materials), the purified genomic DNA should have a 260/280 (nm) ratio &#62;1.8 and a 260/230 ratio between 2.0 - 2.2 to assure that the sample is free of contaminants. The amount of genomic DNA required for NS ranges from 200 ng to 2 &#181;g.
1. Rapid Library Preparation Method (Short Protocol)
NOTE: This is a short protocol. See Table of Materials for the rapid sequencing kit.
<ol>
	<li>In a 0.2 mL thin-walled tube (see Table of Materials) add 200 ng of high molecular weight DNA in a final of volume of 7.5 &#181;L of distilled water.</li>
	<li>Add 2.5 &#181;L of fragmentation mix (FM) from the rapid library preparation kit and mix gently by inversion. Pulse centrifuge to spin down (1,000 x g for 5 s).</li>
	<li>Place the sample in a thermocycler (see Table of Materials) for one round at 30 &#176;C for 1 min followed by 75 &#176;C for 1 min. Remove the tube and pulse centrifuge to spin down the sample.</li>
	<li>Add 1 &#181;L of rapid adapter and 0.2 &#181;L of blunt/TA ligase master mix, and then store the adapted and tethered library on ice. 
	NOTE: If using the short protocol proceed to step 8</li>
</ol>
2. Ligation Sequencing Protocol (Long Protocol): Metagenomic DNA Fragmentation
Note: See Table of Materials for the ligation sequencing kit.
<ol>
	<li>Dilute 1 &#181;g of purified genomic DNA to a final volume of 46 &#181;L in deionized water.</li>
	<li>Transfer the sample to a DNA fragmentation tube (see Table of Materials) and centrifuge for 1 min at room temperature at 8,000 x g using a microcentrifuge (see Table of Materials) to produce ~8 kb genomic fragments. Make sure all the liquid has passed into the collection tube.</li>
	<li>Invert the fragmentation tube, return to the centrifuge, and centrifuge for 1 min to collect the fragmented genomic DNA into the lower chamber.</li>
	<li>Transfer the fragmented genomic DNA to a sterile low-DNA binding 1.5 mL tube, and analyze a portion on a low percentage agarose gel (0.6%) to confirm fragmentation to &#62;30 kb.</li>
</ol>
3. Fragmented Genomic DNA End-preparation
<ol>
	<li>Using 800 ng of fragmented genomic DNA in 45 &#181;L of deionized water, add 7 &#181;L end-prep reaction buffer (see Table of Materials), 3 &#181;L enzyme mix (see Table of Materials), and 5 &#181;L of nuclease free water.</li>
	<li>Mix by inversion and pulse centrifuge (1,000 x g for 5 s).</li>
	<li>Transfer sample to a 0.2 mL thin-walled tube, and incubate sample for 5 min at 20 &#176;C, followed by incubation at 65 &#176;C for 5 min.</li>
	<li>Pulse centrifuge to bring contents to the bottom of the tube.</li>
	<li>Transfer sample to a 1.5 mL low-DNA binding microcentrifuge tube.</li>
	<li>Prepare magnetic beads (see table of materials). Add 60 &#181;L of resuspended beads to the end prep reaction from step 3.5 and mix by pipetting.</li>
	<li>Incubate on a rotator mixer at 100 rpm (see Table of Materials) for 5 min at room temperature.</li>
	<li>Pulse centrifuge the sample to pellet the beads, then place the sample next to magnet.</li>
	<li>Once the beads are adhered to the magnet, pipette off supernatant and discard.</li>
	<li>Remove the tube from the magnet and wash beads with 200 &#181;L of freshly prepared 70% ethanol by gently pipetting the sample up-and-down.</li>
	<li>Pulse centrifuge to pellet the beads and return to the magnet.</li>
	<li>Remove the supernatant and discard. Repeat the washing step again using 200 &#181;L of 70% ethanol.</li>
	<li>Pulse centrifuge the tube, return to the magnet and remove all the 70% ethanol wash.</li>
	<li>With the microcentrifuge tube lid open, allow the beads to air dry for 5 min at room temperature.</li>
	<li>Remove the tube from magnet and suspend the pellet in 31 &#181;L of sterile, nuclease-free water. Incubate for 2 min at room temperature.</li>
	<li>Return the tube to the magnet until all the beads have pelleted and the eluate is clear.</li>
	<li>Transfer the eluate to a 1.5 mL microcentrifuge tube. Using 1 &#181;L of sample, quantify the end-prepped DNA using a UV spectrophotometer at 260 nm (see Table of Materials). 
	Note: The percent recovery should be ~70% (700 ng) from a starting concentration of 1 &#181;g of genomic DNA.</li>
</ol>
4. Adapter and Tether Addition to End-prepped Genomic DNA Fragments
<ol>
	<li>Mix all the blunt/TA ligase master mix tubes by inversion, and then pulse centrifuge to bring contents to the bottom.</li>
	<li>Using 30 &#181;L of end-prepped DNA, add 20 &#181;L of adapter mix, and 50 &#181;L of blunt/TA ligation master mix.</li>
	<li>Mix by inversion, pulse centrifuge, and incubate for 10 min at room temperature.</li>
</ol>
5. Magnetic Bead Preparation
<ol>
	<li>Vortex the beads and then transfer 50 &#181;L of suspended beads to a 1.5 mL microfuge tube.</li>
	<li>Place the tube on the magnet to pellet the beads until the eluate clears.</li>
	<li>Remove the supernatant and discard.</li>
	<li>Add 100 &#181;L of bead binding buffer (see Table of Materials for bead kit) to the beads, vortex to resuspend the beads, pellet the beads on the magnet, remove the supernatant and discard. Repeat the washing with 100 &#181;L of binding buffer.</li>
	<li>Add 100 &#181;L of bead binding buffer to the washed beads. Vortex to resuspend the beads.</li>
</ol>
6. Library Purification
<ol>
	<li>Using a P200 micropipette, add 40 &#181;L of beads from the previous step to the adapted/tethered genomic DNA. Mix the sample carefully by pipetting.</li>
	<li>Incubate the sample on a rotator mixer at 100 rpm for 5 min at room temperature.</li>
	<li>Place the sample on the magnet and allow the beads to settle. Pipette off the supernatant and discard.</li>
	<li>Resuspend the beads in 140 &#181;L of bead binding buffer by pipetting. Place the sample against the magnet, discard the supernatant, and wash again with another 140 &#181;L of bead binding buffer.</li>
	<li>Pulse centrifuge the tube, place the tube on the magnet for 2 min. Remove the remaining bead binding buffer from the pellet.</li>
</ol>
7. Elution of Library from Magnet Beads
<ol>
	<li>Resuspend the magnetic beads in 15 &#181;L of elution buffer by pipetting. Incubate the sample for 10 min at room temperature.</li>
	<li>Place the tube against the magnet to pellet the beads. When the solution clears, remove 15 &#181;L of eluate and transfer to a 1.5 mL microfuge tube, and place the sample on ice.</li>
	<li>Quantify the library using an UV spectrophotometer. 
	NOTE: The percent recovery should be ~25% (250 ng) from a starting concentration of 1 &#181;g of genomic DNA.</li>
</ol>
8. Starting a Run/Quality Control
<ol>
	<li>Remove the flow cell from packaging and attach the flow cell to the portable, real-time sequencer (see Table of Materials).</li>
	<li>Attach the sequencer to a computer via USB cable and start the sequencing software (see Table of Materials).</li>
	<li>Click &#34;Connect&#34; device through the sequencing software, select &#34;NC_Platform_QC.py&#34;, and click &#34;Start&#34;.</li>
	<li>The quality control (QC) takes approximately 6 - 7 min to complete; look for a &#39;sea of green&#39; (large areas of output) on the QC readout to confirm that there are enough active pores (&#62;800) for DNA sequencing.</li>
</ol>
9. Starting a Sequencing Run
<ol>
	<li>Open the sequencing program; a dialog box will appear. In the Sample ID box, name the sample.</li>
	<li>Click on the &#34;Flow Cell ID&#34; box and enter the code found on the sticker on the top of the flow cell.</li>
	<li>Open the flow cell lid and then slide the sample port cover to the right (clockwise) so that the sample port is visible. Once the port is open, check for a small bubble. Remove a few microliters of buffer including the bubble.</li>
	<li>Check to be sure that there is buffer across the sensor array.Make the priming buffer by mixing 480 &#181;L of running buffer with fuel mix (RBF-1, see the Table of Materials) (be sure to mix thoroughly first) with 520 &#181;L nuclease-free water.</li>
	<li>Add 800 &#181;L of the priming buffer to the priming port. Carefully lift the &#34;SpotOn&#34; cover to make it accessible.</li>
	<li>After 5 min, add an additional 200 &#181;L of priming buffer to the priming port.</li>
</ol>
10. Loading the Library
<ol>
	<li>Before preparing the library place the following reagents on ice: RBF-1 (from the sequencing kit), adapted and tethered library, and library loading beads (LLB; from the sequencing kit).</li>
	<li>To a 0.2 mL centrifuge tube, add 25.5 &#181;L of RBF and 12 &#181;L of nuclease-free water kept at room temperature.</li>
	<li>Mix the LLB by pipetting up-and-down. Add 26.5 &#181;L of the LLB to the 0.2 &#181;L tube. Mix the reagents by pipetting up-and-down. 
	NOTE: The LLB tends to settle out of the mixture.</li>
	<li>Add 11 &#181;L of the adapted and tethered library to the 0.2 &#181;L tube. Mix by inversion and spin down by pulse centrifugation.</li>
	<li>Add 75 &#181;L of sample from step 9.4 to the &#34;SpotOn&#34; port dropwise. Make sure that each drop flows into the port before adding the next.</li>
	<li>Carefully replace the sample port cover so that the bung enters the port, and replace the device lid.</li>
</ol>
11. Starting a Sequencing Software Protocol Script
<ol>
	<li>Open the dropdown menu under &#39;Select Program&#39; and select &#34;48 hour sequencing.&#34;</li>
	<li>Click on the &#34;Execute&#34; button to start the script.</li>
	<li>Check the results of the Mux scan reported in the notification stream for the number of active pores in the MUX scan. 
	&#8203;NOTE: This number may differ from the number of pores in the QC run and a large difference may indicate a problem.
	<ol>
		<li>If there is a significant reduction of active pores from the QC run, restart the software, and if there is still a significant difference, reboot the computer.</li>
	</ol>
	</li>
	<li>Check that the heatsink temperature is 34 &#176;C as reported by the sequencing software script. 
	NOTE: If the temperature is not at 34 &#176;C then there will not be enough active pores for the run.</li>
	<li>Check the histogram of read lengths to determine if the sequence lengths are expected and appropriate for the experimental design.</li>
	<li>Close the desktop agent using &#39;close x.&#39;&#160;Quit the sequencing software by closing down the web GUI. Disconnect the device from the computer.</li>
</ol>
12. Analysis of Run and Results
Note: During the sequencing run, the data can be monitored using the &#34;VIEW REPORT&#34; program using the desktop agent. During or after the run is complete, files are available in a FAST5 file format in the data &#8594; reads &#8594;&#160;pass folder (default). If this folder is open while sequencing is active, the computer may freeze.
<ol>
	<li>To view and manipulate sequences, download a &#34;HDFViewer&#34; .</li>
	<li>To view the sequencing files, directly open the HDF viewer &#8594;&#160;open Data folder &#8594;&#160;select All file types &#8594;&#160;select C: drive (default) &#8594;&#160;select data &#8594;&#160;select reads &#8594;&#160;select pass.</li>
	<li>Find and select the FASTQ file in the left dropdown menu; files will open and can be saved in FASTQ format. 
	NOTE: For the purpose of the student experiment, sequences of 350 nucleotides or longer were selected for further analysis. Output from the base calling allows the user to sort sequence reads based on size. FASTQ files can be analyzed via BLAST (NCBI) or other bioinformatics programs.</li>
</ol>

<ol><li>Quick, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time%2C+portable+genome+sequencing+for+Ebola+surveillance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Real-time, portable genome sequencing for Ebola surveillance.</a> Nature. 530 (7589), 228-232 (2016).</li>
<li>Greninger, A. L., et al. <a target="_blank" href="https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-015-0220-9">Rapid metagenomics identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis.</a> Genome Med. 7, 99(2015).</li>
<li>Quick, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+draft+sequencing+and+real-time+nanopore+sequencing+in+a+hospital+outbreak+of+Salmonella%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella.</a> Genome Biology. 16, 114(2015).</li>
<li>Ashton, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MinION+nanopore+sequencing+identifies+the+position+and+structure+of+a+bacterial+antibiotic+resistance+island%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island.</a> Nat Biotechnol. 33, 296-300 (2015).</li>
<li>Loman, N. J., Quick, J., Simpson, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+complete+bacterial+genome+assembled+de+novo.+using+only+nanopore+sequencing+data%5BTitle%5D)+AND+%22Nat+Method%22%5BJournal%5D)">A complete bacterial genome assembled de novo. using only nanopore sequencing data.</a> Nat Method. 12, 733-735 (2015).</li>
<li>Hoenen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanopore+Sequencing+as+a+Rapidly+Deployable+Outbreak+Tool%5BTitle%5D)+AND+%22Emerg+Infect+Dis%22%5BJournal%5D)">Nanopore Sequencing as a Rapidly Deployable Outbreak Tool.</a> Emerg Infect Dis. 22 (2), 331-334 (2016).</li>
<li>Petrosino, J. F., Highlander, S., Luna, R. A., Gibbs, R. A., Versalovic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metagenomic+pyrosequencing+and+microbial+identification%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">Metagenomic pyrosequencing and microbial identification.</a> Clin Chem. 55 (5), 856-866 (2009).</li>
<li>Cummings, P. J., Ahmed, R., Durocher, J. A., Jessen, A., Vardi, T., Obom, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyrosequencing+for+Microbial+Identification+and+Characterization%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Pyrosequencing for Microbial Identification and Characterization.</a> J. Vis. Exp. (78), e50405(2013).</li>
<li>Rothberg, J. W., et al. <a target="_blank" href="http://www.nature.com/nature/journal/v475/n7356/full/nature10242.html?foxtrotcallback=true">An integrated semi-conductor device that enabling non-optical genome sequencing.</a> Nature. 475, 348-352 (2011).</li>
<li>Levene, M. J., Korlach, J., Turner, S. W., Foquet, M., Craighead, H. G., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zero-mode+Waveguides+for+Single-Molecule+Analysis+at+high+concentrations%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Zero-mode Waveguides for Single-Molecule Analysis at high concentrations.</a> Science. 299, 682-686 (2003).</li>
<li>Eid, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-Time+DNA+Sequencing+from+Single+Polymerase+Molecules%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Real-Time DNA Sequencing from Single Polymerase Molecules.</a> Science. 323, 133-138 (2009).</li>
<li>Stoddart, D., Heron, A. J., Mikhailova, E., Maglia, G., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-nucleotide+discrimination+in+immobilized+DNA+oligonucleotides+with+a+biological+nanopore%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore.</a> PNAS. 106, 7702-7707 (2009).</li>
<li>Shendure, J., Hanlee, J. Next generation DNA sequencing. Nat Biotechnol. 26, 1135-1145 (2008).</li>
<li>Daniel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Metagenomics+of+Soil%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">The Metagenomics of Soil.</a> Nat Rev Microbiol. 3, 470-478 (2005).</li>
</ol>

The authors have no disclosures.

Many next generation sequencing methods have been generated and each depends on sequencing by synthesis but the detection platforms for identifying nucleotides differ. NS, the most recent addition to the marketplace, uses a different method entirely, which does not require a sequencing reaction or labeled nucleotides. This method takes advantage of charge differential of already incorporated nucleotides as they pass through an electrified pore. The identification of each nucleotide occurs by modulation of the electrical current by the different bases as they pass through the nanopore. This multiplexed system allows the user to sequence many fragments at a time. By sequencing both strands of the DNA, the accuracy of the sequence is significantly increased and the sequencing software, which can be loaded onto a laptop computer, processes the signals and provides the sequence information that can be analyzed.
In pyrosequencing, a sequencing by synthesis (SBS) method, detection of the specific base incorporated into the template depends on the luciferase assay and the generation of chemiluminescent signals8. In ion semiconductor sequencing, the released hydrogen ion, which decreases the pH is detected by an ion sensor9. Single molecule real-time sequencing depends on the zero mode wave guide (ZMW)10, which illuminates for detection a florescent molecule tagged to the incorporated nucleotide. SBS uses a unique method to amplify the target DNA such that clusters of unique sequences are generated13. Detection of the added nucleotide is achieved when the fluorescence of the tagged nucleotide is recorded. NS on the other hand has unique advantage over other methods in that it requires limited technical resources, is portable, produces long sequencing reads, requires no prior DNA amplification, and can be operated at a reduced cost compared to other methods. Our students found the newer, rapid library prep protocol to be straightforward and amenable to a three hour lab class. Some of the issues we encountered were bubbles in the flow cell which were difficult to remove, it required significant computer power (one terabyte of storage), the current output of data is in a FAST5 file, and the sequencing flow cell has a limited shelf life before it deteriorates. In addition, other disadvantages of the NS Ligation sequencing protocol (long protocol) is that it requires several library preparation steps, requires expertise in molecular biology techniques, and generates reduced sequencing fidelity when compared to some sequencing methodologies3. However, recent advances with the new rapid library preparation kit requires only 10 min for library preparation and has demonstrated a reduced sequencing error rate. The new library preparation method was very amenable for use in a lab class.
There are several critical steps in the protocol, particularly in the QC of the flow cell. This includes performing an initial QC within five days of the receipt of the flow cell and using them within 8 weeks. Although we have used flow cells that were beyond 8 weeks, the number of open/active pores is greatly reduced. It is important that experiments are planned to fit a timeline where maximum use of the flow cells is achieved. We have used the cleaning protocol and reused the flow cells with success.
Investigating the metagenomics of soil represent an untapped genetic reservoir of microbial diversity. For example, one gram of soil is estimated to contain between 107&#160;- 109 prokaryotic cells14. Moreover, soil organisms are a main source of novel natural products, enzymes, and antibiotics. Thus, soil metagenomics DNA sequence analysis represents a valuable instructional tool for students at every level of education. The NS technology&#39;s ease of use and low cost make this system a very effective teaching tool. Students can sequence environmental samples, and upon completion of the sequence use available bioinformatics tools to identify and characterize microbes and metagenomics sequences in test samples. Using the NS technology, students have true hands-on experience, which has until now been out of reach for use in laboratory courses because of the advanced technical expertise and high reagent, equipment, and maintenance costs in other sequencing platforms. Recently, one of our students (J. Harrison, personal communication) reported the use of this technology in an environmental monitoring project of farm soils. We expect that there will be many more applications for this technology in the education space.

The goal of this procedure is to demonstrate the steps required for preparation of an environmental DNA library for sequencing, utilization of a nanopore flow cell sequencing device, and to perform analysis of the generated DNA sequences using system software and the National Center for Biotechnology Information (NCBI) bioinformatics tools to identify microbial species in soil. Currently, most DNA sequencing platforms require a major investment in technical training and complex instrumentation, which is not feasible in resource poor environments or in field applications. The nanopore sequencing (NS) platform eliminates these issues with a cost effective, simple to use library preparation protocol, and a portable device to sequence and analyze a variety of different types of nucleic acids1,3. We have incorporated the NS platform into several lab classes for master&#39;s degree students.
Nanopore technology for sequencing biomolecules has demonstrated wide applications in the life sciences, including identification of bacterial and viral pathogens1,2,6, environmental biodiversity studies, food safety monitoring, genomic analysis3,5, and characterization of bacterial antibiotic resistance4. NS is a fast and accurate method to sequence nucleic acids that is based on the principle of &#34;strand sequencing&#34; by detecting electrical disruption by individual nucleotide bases when single stranded DNA passes through a nanopore inserted into an electrified synthetic polymer membrane. The steps involved in preparing DNA for NS include genome fragmentation, end repair and 3&#39; dA-tailing of genomic fragments, adaptor and tether annealing to the DNA, DNA library purification, and loading the library into the nanopore flow cell device. Fragmentation of the genome into ~8 kb sizes is accomplished by centrifuging 1 - 2 &#181;g of genomic DNA through a g-tube fragmentation tube. The fragmented genomic ends are then repaired and tailed with poly dA using a commercially available kit. Single stranded adapter sequences, which are compatible with the nanopore motor protein, are added to DNA ends which are used to guide the DNA sequence through the nanopore (Figure 1). The tether sequences are required for DNA purification and for localizing the DNA molecules to the pore membrane. The hairpin is generated by ligating a hairpin adapter to one end of the dA tailed library. The hairpin structures at the DNA ends allows reading of the sense and antisense strands as the DNA passes through the nanopore (Figure 2). The prepared genomic library is then purified from the reaction by using streptavidin beads using a magnetic field, followed by loading the sample into the nanopore flow cell for analysis.
The sequenced DNA is assessed for quality and sequencing reads that are acceptable for analysis are then subjected to several bioinformatics tools to identify microbes. The sequences are &#34;translated&#34; into a FASTQ from a FAST5 format. In the FASTQ format, the sequences can then be used in BLAST analysis.

<table><tbody><tr><td>Thermal Cycler</td><td>LifeECO</td><td>BTC42096</td><td></td></tr><tr><td>Covaris g-TUBE</td><td>Covaris</td><td>520079</td><td></td></tr><tr><td>NEBNext End Repair Module</td><td>New England BioLab</td><td>E7546</td><td></td></tr><tr><td>Eppendorf LoBind Centrifuge Tubes</td><td>Sigma-Aldrich</td><td>Z666505</td><td></td></tr><tr><td>NEB Blunt/TA Ligase Master Mix</td><td>New England BioLab</td><td>MO367</td><td></td></tr><tr><td>MyOne C1 Strepavidin Beads</td><td>Thermo Fisher</td><td>65001</td><td></td></tr><tr><td>Eppendorf microcentrifuge</td><td>Eppendorf</td><td></td><td>Model 5420</td></tr><tr><td>Nanodrop UV spectrophotometer</td><td>Thermo Fisher</td><td>ND-2000</td><td>Model 2000</td></tr><tr><td>Belly Dancer Orbital Shaker</td><td>Sigma-Aldrich</td><td>Z768499</td><td></td></tr><tr><td>Power Soil DNA Isolation Kit</td><td>MO BIO</td><td>12888-50</td><td></td></tr><tr><td>Ligation Sequencing Kit 2D</td><td>Oxford Nanopore&amp;nbsp;</td><td>SQK-LSK208</td><td></td></tr><tr><td>Rapid Sequencing Kit for Genomic DNA</td><td>Oxford Nanopore&amp;nbsp;</td><td>SQK-RAD002</td><td></td></tr><tr><td>End-Prep Reaction Buffer and enzyme mix (NEB Blunt/TA Ligase Naster Mix)</td><td>New England BioLab</td><td>MO367</td><td></td></tr><tr><td>AmPure XP Magnetic Beads</td><td>Beckman Coulter</td><td>A63880</td><td></td></tr><tr><td>Basic Starter Pack&amp;nbsp;</td><td>Oxford Nanopore&amp;nbsp;</td><td></td><td>Includes MinION&amp;nbsp; Sequencing Device and Flow Cell</td></tr><tr><td>MinKNOW software</td><td>Oxford Nanopore&amp;nbsp;</td><td></td><td></td></tr><tr><td>Rapid Sequencing Kit for Genomic DNA</td><td>Oxford Nanopore&amp;nbsp;</td><td>SQK-RAD002</td><td>Includes Running Buffer with Fuel (RBF), Fragmentation Mix (FRM) Rapid Adapter (RAD) Library Lodaing Beads (LLB)</td></tr></tbody></table>

nanopore dna sequencing for metagenomic soil analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

The experimental design and nanopore technology provided a fast and inexpensive method for students to sequence soil DNA. The representative run passed the quality control parameters with more than 800 pores available for sequencing. The run resulted in over 125,000 reads available for study with a median sequence length of 5.38 kb. Sequences are given a quality score and only those sequences with acceptable scores were then analyzed. As the output of the sequencing reaction is in FAST5 format, which is not accepted by programs such as BLAST (NCBI), the sequences were viewed in the HDF viewer which converts the sequences to FASTQ format, which is compatible for BLAST analysis. In future iterations of the software, the data will be available as a FASTQ format, eliminating the need for the HDF viewer. See Supplementary Figures 1, 2, and 3. 
Fifty sequences of over 350 nucleotides in length were subject to analysis by BLASTN (nucleotides against nucleotide database) or BLASTX (nucleotides translated into amino acid sequence) (NCBI) to identify organisms in the soil sample. Given the time constraints of the class and that the focus of the course is on laboratory methods and not bioinformatics, we have the students analyze sequences within these parameters. Our bioinformatics classes can use the data for development of pipeline analysis tools. This level of bioinformatics analysis is not covered in the laboratory based class. Table 1 is a list of the organisms that were identified with e values of less than 4 e-04. Generally, e-values less than 4 e-04 indicate strong similarity between the input sequence (query) and the matched sequence. The organisms identified by BLASTN (against the non-redundant (nr) database) are from a wide variety of species and are available for further genomic and protein analyses. This sample of soil, which came from a garden in Baltimore County, MD had never been tested, so there were no previous data from which to determine what organisms might be in the soil. BLASTN analysis (against the nr database) analysis also indicated there were sequences with no similarities in the nr database. To determine if these are truly novel sequences, further study is needed. Analysis of several sequences by BLASTX revealed several proteins from organisms not represented in the BLASTN analysis. Table 2 lists several possible proteins including endopeptidases, and ATPases-like proteins. Using this library of sequences, students have the opportunity to use more sophisticated bioinformatics tools to perform further analysis, if directed by the instructor.
<img alt="Nanopore sequencing kit diagram; DNA prep, adapter ligation, tethering for sequencing analysis." class="xfigimg" src="/files/ftp_upload/55979/55979fig1.jpg" data-altupdatedat="1747929244000" data-alt="Nanopore sequencing kit diagram: DNA preparation, end-repair, adapter ligation, tether attachment."> 
Figure 1: Genomic library preparation. Steps for preparation of genomic DNA library for sequencing using nanopore technology. This figure has been modified with the necessary permissions. <a href="https://cloudflare.jove.com/files/ftp_upload/55979/55979fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="DNA sequencing process diagram with polymerase enzyme and nucleotide data output." class="xfigimg" src="/files/ftp_upload/55979/55979fig2.jpg" data-altupdatedat="1747929244000" data-alt="RNA transcription process, DNA to RNA translation, gene expression diagram, molecular biology concepts."> 
Figure 2. Schematic of DNA sequencing using a nanopore. DNA passing through a nanopore membrane with electrical signal generation and base identification. This figure has been modified with the necessary permissions. <a href="https://cloudflare.jove.com/files/ftp_upload/55979/55979fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Organism</td>
			<td>e value</td>
		</tr>
		<tr>
			<td>Streptomyces sp.</td>
			<td>3.00E-06</td>
		</tr>
		<tr>
			<td>Nocardoides sp.</td>
			<td>5.00E-04</td>
		</tr>
		<tr>
			<td>Gordonia sp.</td>
			<td>5.00E-04</td>
		</tr>
		<tr>
			<td>Hyphomicrobium nitrativorens</td>
			<td>1.00E-139</td>
		</tr>
		<tr>
			<td>Starkeya novella</td>
			<td>1.00E-31</td>
		</tr>
		<tr>
			<td>Hyphomicrobium denitrificans</td>
			<td>1.00E-30</td>
		</tr>
		<tr>
			<td>Fibomicrobium sp.</td>
			<td>5.00E-17</td>
		</tr>
		<tr>
			<td>Pseudomonas sp.</td>
			<td>2.00E-15</td>
		</tr>
		<tr>
			<td>Rhodothemus marinus</td>
			<td>1.00E-17</td>
		</tr>
		<tr>
			<td>Turneiella parva</td>
			<td>2.00E-24</td>
		</tr>
		<tr>
			<td>E. coli</td>
			<td>0.00E+00</td>
		</tr>
		<tr>
			<td>Bradyrhizobium sp.</td>
			<td>3.00E-30</td>
		</tr>
		<tr>
			<td>Gemmatirosa kalamazoonesis</td>
			<td>1.00E-20</td>
		</tr>
		<tr>
			<td>Burkholderia sp.</td>
			<td>8.00E-10</td>
		</tr>
		<tr>
			<td>Sphingomonas sp.</td>
			<td>8.00E-10</td>
		</tr>
		<tr>
			<td>Cellumonas sp.</td>
			<td>6.00E-11</td>
		</tr>
	</tbody>
</table>
Table 1: Selected results of BLASTN analysis. Soil metagenomics DNA sequences subjected to BLASTN similarity search against the NCBI non-redundant nucleotide database. The data reported have e-values of 4 x e-4 or less.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Protein </td>
			<td>Organism</td>
		</tr>
		<tr>
			<td>Hypothetical protein </td>
			<td>Acidobacter bacterium</td>
		</tr>
		<tr>
			<td>SAM dependent methyltransferase  </td>
			<td>H. denitrificans</td>
		</tr>
		<tr>
			<td>ATPase </td>
			<td> Jannaschia sp.</td>
		</tr>
		<tr>
			<td>Endopeptidase </td>
			<td>Shigella sonnei</td>
		</tr>
		<tr>
			<td>Lysis protein </td>
			<td>E. coli</td>
		</tr>
	</tbody>
</table>
Table 2: Selected results of BLASTX analysis. Soil metagenomics DNA sequences subjected to BLASTX similarity search against the NCBI non-redundant protein database.
<img alt="Nanopore sequencing dashboard with MinKNOW software interface showing live data, physical layout, pore status, and notifications." src="/files/ftp_upload/55979/55979supfig1.jpg" data-altupdatedat="1747929244000" data-alt="DNA sequencing process using MinION software, diagram with data visualization and notification panel."> 
Supplemental Figure 1: Platform QC results. The results of the platform QC are presented. In the upper right corner are the results of the QC for active pores. Each quadrant of the flow cell is tested for active pores. The main page is dynamic and changes as each quadrant is tested. In this run, 1,414 single pores were detected. <a href="https://cloudflare.jove.com/files/ftp_upload/55979/55979supfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="DNA sequence data analysis using HDFView software; file explorer with sequencing result files displayed." src="/files/ftp_upload/55979/55979supfig2.jpg" data-altupdatedat="1747929244000" data-alt="DNA sequencing analysis interface with data files and sequence alignment display."> 
Supplemental Figure 2: Converting FAST5 files to FASTQ files. Here on the right side, is the output of the data from the sequencing run. Each line represents an individual sequence. The left side of the figure shows the highlighted sequence from the right side, in the HDF viewer that converts the sequence to a FASTQ file which can be used for further analysis. <a href="https://cloudflare.jove.com/files/ftp_upload/55979/55979supfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="DNA sequencing analysis with HPFview software displaying nucleotide sequences; bioinformatics." src="/files/ftp_upload/55979/55979supfig3.jpg" data-altupdatedat="1747929244000" data-alt="DNA sequencing data in software interface; genetic analysis, bioinformatics tool demonstration."> 
Supplemental Figure 3: Example of FASTQ sequence in HDF viewer. This sequence (read 803) is in a FASTQ file which converts the FAST5 data into nucleotides. <a href="https://cloudflare.jove.com/files/ftp_upload/55979/55979supfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Nanopore DNA Sequencing for Metagenomic Soil Analysis at JoVE.com

Nanopore DNA Sequencing for Metagenomic Soil Analysis

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

nanopore-dna-sequencing-for-metagenomic-soil-analysis

Research

JoVE Journal

Environment

30.5K Views.  Johns Hopkins University. Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

Video: Nanopore DNA Sequencing for Metagenomic Soil Analysis

Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. 
In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Metagenomics was used to investigate the microbiome of silage cattle feed. Analysis was performed by shotgun sequencing. This approach was used to characterize the composition of the microbial community within the cattle feed.

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of ...

Genetics

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.

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 ...

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 ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Chemistry

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Biology

Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

The protocol detects key methane-cycling genes in South Texas coastal wetlands and visualizes their spatial distribution to enhance understanding of methane regulation and its environmental impacts in these dynamic ecosystems.

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

Bioengineering

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...