The overall goal of this video is to demonstrate how to sequence metagenomic soil DNA using a nanopore sequencing methodology. This method can help educators introduce DNA sequencing into a classroom setting and provide real-time generation of novel DNA sequencing data for analysis. The main advantage of this technique is that it is fast, affordable, and requires minimal laboratory equipment.
This technique also gives students the opportunity to use more sophisticated bioinformatics tools that are usually available in laboratory classes. This short protocol works with a rapid sequencing kit. To begin add 200 nanograms of high molecular weight DNA into a 0.2 milliliter thin-walled tube and top off the volume with distilled water to 7.5 microliters.
Next add 2.5 microliters of fragmentation mix from the kit and mix the tube gently using inversions. Then use a pulse of centrifugation to spin down the tubes contents. Now transfer the tube to a thermocycler for a one minute cycle at 30 degrees Celsius, followed by a second one minute cycle at 75 degrees Celsius.
Then pulse-spin the tube again. To complete the adapted and tethered library add one microliter of rapid adapter and 0.2 microliters of blunt/TA ligase master mix. Then use inversion to mix the tube, followed by another pulse spin and store the tube on ice.
First, set-up the sequencer, remove the Flow Cell from its packaging and attach it to the portable real-time sequencer. Then attach the sequencer to a computer using the USB cable and start the sequencing software. In the software select the start device command and chose the option for QC platform.
Then press execute to run the the quality control. After about seven minutes the quality control run will be complete. The green highlighting on the readout indicates that there are active pores available for DNA sequencing.
At least 800 active pores are required. Now go to the dialogue box. In the Sample ID field enter the name of the sample to be sequenced.
For the Flow Cell ID enter the code found on the sticker attached to the top of the Flow Cell. Now open the sequencer lid and then slide the priming port cover to the left to access the port. Now remove a few microliters of buffer, including a small bubble that is often present.
Now make enough buffer to fill the sensor array. Prepare one milliliter of freshly made thoroughly mixed priming buffer. Then add 800 microliters of priming buffer and wait five minutes.
After five minutes add the remaining 200 microliters of priming buffer. To prepare the library for loading chill RBF1 and LLB on ice along with the adapted and tethered library. Then to new 0.2 milliliter centrifuge tube add 25.5 microliters of RBF1 and 12 microliters of room temperature nucleus-free water.
Next mix the LLB with pipette trituration then take a 26.5 microliter aliquot and add it to the tube with diluted RBF1. Mix the reagents using more trituration to prevent the beads within from settling. Now add 11 microliters of the adapted and tethered library and mix the tube gently using inversion.
Then use a pulse spin to pool the sample. While avoiding the beads take 75 microliters of the sample and add it to the spot on port drop wise. Make sure that each drop flows into the port before adding the next.
When loading the sample it is vital that each drop enters the port before the next is added. It is also important to avoid resuspending the beads when loading the sample port which can clog the port. Now replace the sample port cover making certain that the bung enters the port.
Then replace the device lid and proceed with executing the 48 hour sequencing run option selected through the software. Using the described procedure provide a fast and inexpensive method for sequencing soil DNA for educational purposes. Runs typically yielded over 125, 000 reads which averaged over 5, 000 base pairs.
Each run was given a quality score to select those most useful for further analysis. Next each students subjected 50 sequences of at least 350 nucleotides to BLASTN or BLASTX analysis to identify organism from the sample. Identified organism with e-values less than 0.0004 are considered strong matches.
Many sequences subjected to BLASTN where unmatched and made relate to novel organisms. BLASTX revealed several proteins from organisms not represented in the BLASTN analysis. Of interest Endopeptidase and ATPase like proteins where identified.
After watching this video you should have a good understanding of how to prepare and analyze soil DNA for sequencing using nanopore sequencing technology. This protocol can be completed in two and a half hours, requires minimal laboratory equipment or technical expertise and can be completed in a classroom setting. While attempting and completing this procedure it's important to remember to pipe the reagents carefully, to mix samples thoroughly and to centrifuge samples between steps to avoid losing sample.
