Nanopore DNA Sequencing for Metagenomic Soil Analysis

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 >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 µg.

1. Rapid Library Preparation Method (Short Protocol)

NOTE: This is a short protocol. See Table of Materials for the rapid sequencing kit.

1. 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 µL of distilled water.
2. Add 2.5 µL 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).
3. Place the sample in a thermocycler (see Table of Materials) for one round at 30 °C for 1 min followed by 75 °C for 1 min. Remove the tube and pulse centrifuge to spin down the sample.
4. Add 1 µL of rapid adapter and 0.2 µL 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

2. Ligation Sequencing Protocol (Long Protocol): Metagenomic DNA Fragmentation

Note: See Table of Materials for the ligation sequencing kit.

1. Dilute 1 µg of purified genomic DNA to a final volume of 46 µL in deionized water.
2. 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.
3. Invert the fragmentation tube, return to the centrifuge, and centrifuge for 1 min to collect the fragmented genomic DNA into the lower chamber.
4. 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 >30 kb.

3. Fragmented Genomic DNA End-preparation

1. Using 800 ng of fragmented genomic DNA in 45 µL of deionized water, add 7 µL end-prep reaction buffer (see Table of Materials), 3 µL enzyme mix (see Table of Materials), and 5 µL of nuclease free water.
2. Mix by inversion and pulse centrifuge (1,000 x g for 5 s).
3. Transfer sample to a 0.2 mL thin-walled tube, and incubate sample for 5 min at 20 °C, followed by incubation at 65 °C for 5 min.
4. Pulse centrifuge to bring contents to the bottom of the tube.
5. Transfer sample to a 1.5 mL low-DNA binding microcentrifuge tube.
6. Prepare magnetic beads (see table of materials). Add 60 µL of resuspended beads to the end prep reaction from step 3.5 and mix by pipetting.
7. Incubate on a rotator mixer at 100 rpm (see Table of Materials) for 5 min at room temperature.
8. Pulse centrifuge the sample to pellet the beads, then place the sample next to magnet.
9. Once the beads are adhered to the magnet, pipette off supernatant and discard.
10. Remove the tube from the magnet and wash beads with 200 µL of freshly prepared 70% ethanol by gently pipetting the sample up-and-down.
11. Pulse centrifuge to pellet the beads and return to the magnet.
12. Remove the supernatant and discard. Repeat the washing step again using 200 µL of 70% ethanol.
13. Pulse centrifuge the tube, return to the magnet and remove all the 70% ethanol wash.
14. With the microcentrifuge tube lid open, allow the beads to air dry for 5 min at room temperature.
15. Remove the tube from magnet and suspend the pellet in 31 µL of sterile, nuclease-free water. Incubate for 2 min at room temperature.
16. Return the tube to the magnet until all the beads have pelleted and the eluate is clear.
17. Transfer the eluate to a 1.5 mL microcentrifuge tube. Using 1 µL 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 µg of genomic DNA.

4. Adapter and Tether Addition to End-prepped Genomic DNA Fragments

1. Mix all the blunt/TA ligase master mix tubes by inversion, and then pulse centrifuge to bring contents to the bottom.
2. Using 30 µL of end-prepped DNA, add 20 µL of adapter mix, and 50 µL of blunt/TA ligation master mix.
3. Mix by inversion, pulse centrifuge, and incubate for 10 min at room temperature.

5. Magnetic Bead Preparation

1. Vortex the beads and then transfer 50 µL of suspended beads to a 1.5 mL microfuge tube.
2. Place the tube on the magnet to pellet the beads until the eluate clears.
3. Remove the supernatant and discard.
4. Add 100 µL 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 µL of binding buffer.
5. Add 100 µL of bead binding buffer to the washed beads. Vortex to resuspend the beads.

6. Library Purification

1. Using a P200 micropipette, add 40 µL of beads from the previous step to the adapted/tethered genomic DNA. Mix the sample carefully by pipetting.
2. Incubate the sample on a rotator mixer at 100 rpm for 5 min at room temperature.
3. Place the sample on the magnet and allow the beads to settle. Pipette off the supernatant and discard.
4. Resuspend the beads in 140 µL of bead binding buffer by pipetting. Place the sample against the magnet, discard the supernatant, and wash again with another 140 µL of bead binding buffer.
5. Pulse centrifuge the tube, place the tube on the magnet for 2 min. Remove the remaining bead binding buffer from the pellet.

7. Elution of Library from Magnet Beads

1. Resuspend the magnetic beads in 15 µL of elution buffer by pipetting. Incubate the sample for 10 min at room temperature.
2. Place the tube against the magnet to pellet the beads. When the solution clears, remove 15 µL of eluate and transfer to a 1.5 mL microfuge tube, and place the sample on ice.
3. Quantify the library using an UV spectrophotometer.
   NOTE: The percent recovery should be ~25% (250 ng) from a starting concentration of 1 µg of genomic DNA.

8. Starting a Run/Quality Control

1. Remove the flow cell from packaging and attach the flow cell to the portable, real-time sequencer (see Table of Materials).
2. Attach the sequencer to a computer via USB cable and start the sequencing software (see Table of Materials).
3. Click "Connect" device through the sequencing software, select "NC_Platform_QC.py", and click "Start".
4. The quality control (QC) takes approximately 6 – 7 min to complete; look for a 'sea of green' (large areas of output) on the QC readout to confirm that there are enough active pores (>800) for DNA sequencing.

9. Starting a Sequencing Run

1. Open the sequencing program; a dialog box will appear. In the Sample ID box, name the sample.
2. Click on the "Flow Cell ID" box and enter the code found on the sticker on the top of the flow cell.
3. 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.
4. Check to be sure that there is buffer across the sensor array.Make the priming buffer by mixing 480 µL of running buffer with fuel mix (RBF-1, see the Table of Materials) (be sure to mix thoroughly first) with 520 µL nuclease-free water.
5. Add 800 µL of the priming buffer to the priming port. Carefully lift the "SpotOn" cover to make it accessible.
6. After 5 min, add an additional 200 µL of priming buffer to the priming port.

10. Loading the Library

1. 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).
2. To a 0.2 mL centrifuge tube, add 25.5 µL of RBF and 12 µL of nuclease-free water kept at room temperature.
3. Mix the LLB by pipetting up-and-down. Add 26.5 µL of the LLB to the 0.2 µL tube. Mix the reagents by pipetting up-and-down.
   NOTE: The LLB tends to settle out of the mixture.
4. Add 11 µL of the adapted and tethered library to the 0.2 µL tube. Mix by inversion and spin down by pulse centrifugation.
5. Add 75 µL of sample from step 9.4 to the "SpotOn" port dropwise. Make sure that each drop flows into the port before adding the next.
6. Carefully replace the sample port cover so that the bung enters the port, and replace the device lid.

11. Starting a Sequencing Software Protocol Script

1. Open the dropdown menu under 'Select Program' and select "48 hour sequencing."
2. Click on the "Execute" button to start the script.
3. Check the results of the Mux scan reported in the notification stream for the number of active pores in the MUX scan.
   ​NOTE: This number may differ from the number of pores in the QC run and a large difference may indicate a problem.
   1. 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.
4. Check that the heatsink temperature is 34 °C as reported by the sequencing software script.
   NOTE: If the temperature is not at 34 °C then there will not be enough active pores for the run.
5. Check the histogram of read lengths to determine if the sequence lengths are expected and appropriate for the experimental design.
6. Close the desktop agent using 'close x.' Quit the sequencing software by closing down the web GUI. Disconnect the device from the computer.

12. Analysis of Run and Results

Note: During the sequencing run, the data can be monitored using the "VIEW REPORT" program using the desktop agent. During or after the run is complete, files are available in a FAST5 file format in the data → reads → pass folder (default). If this folder is open while sequencing is active, the computer may freeze.

1. To view and manipulate sequences, download a "HDFViewer" .
2. To view the sequencing files, directly open the HDF viewer → open Data folder → select All file types → select C: drive (default) → select data → select reads → select pass.
3. 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.