Sequencing

Once we had the amplified 16S DNA, we were almost ready to start sequencing!

One cool thing we did in this procedure was to use magnetic separation with magnetic beads to further concentrate the DNA: Figure 1. "GE MagRack 6 pipetting.".

The beads are coated to stick to the DNA, which allows us to easily extract the DNA from the rest of the solution. This is cool, but bio people are borderline obsessed by those beads! Go figure!

Then we prepared the DNA for sequencing with the Oxford Nanopore specific part: Oxford Nanopore SQK-LSK109 Ligation Sequencing Kit.

Here some of the steps required a bit more of vortexing for mixing the reagents, and for this we used the VELP Scientifica WIZARD IR Infrared Vortex Mixer which appears to be quicker to use and not as strong as the Vortex Genie 2 previously used to break up the cells:

After all that was done, the DNA of the several 400 ml water bottles and hours of hard purification labour were contained in one single Eppendorf!

With all this ready, we opened the Nanopore flow cell, which is the 500 dollar consumable piece that goes in the sequencer.

We then had to pipette the final golden Eppendorf into the flow cell. My anxiety levels were going through the roof: Figure 4. "Oxford nanopore MinION flow cell pipette loading.".

At this point bio people start telling lab horror stories of expensive solutions being spilled and people having to recover them from fridge walls, or of how people threw away golden Eppendorfs and had to pick them out of trash bins with hundreds of others looking exactly the same etc. (but also how some discoveries were made like this). This reminded Ciro of: youtu.be/89UNPdNtOoE?t=919 Alfred Maddock's plutonium spill horror story.

Luckily this time, it worked out!

We then just had to connect the MinION to the computer, and wait for 2 days.

During this time, the DNA would be sucked through the pores.

As can be seen from Video 1. "Oxford Nanopore MinION software channels pannel on Mac." the software tells us which pores are still working.

Pores go bad sooner or later randomly, until there are none left, at which point we can stop the process and throw the flow cell away.

48 hours was expected to be a reasonable time until all pores went bad, and so we called it a day, and waited for an email from the PuntSeq team telling us how things went.

We reached a yield of 16 billion base pairs out of the 30Gbp nominal maximum, which the bio people said was not bad.