Week 7: Sequencing!
Welcome back, reader!
This week I started sequencing the angelfish RNA (well… cDNA… read the last post if you want to know the difference).
But before we start talking about the sequencing itself, I think it would be worthwhile to discuss Nanopore sequencing, the sequencing technology I am using to obtain my data. Nanopore sequencing is a marvel of both material science and protein engineering.
It starts with engineered protein nanopores that are embedded in a membrane. DNA is then moved through these nanopores with the help of motor proteins. As DNA moves through these channels, it disrupts an electrical current passing through the membrane. The specific disruption is unique to each nucleotide and the changes in current is recorded. A basecaller on a computer then analyzes the signal current data and determines which nucleotide was passing through the pore at the specific point and time. A nice diagram that shows all of this is shown below.
Okay! Now that we’ve talked about that, let’s look at the start of my sequencing run! I loaded approximately 44 nanograms of the prepared cDNA library into the flow cell. Within an hour, I could see that the sequencing was off to a great start! The nice thing about nanopore sequencing is that it allows you to visualize and understand your data in real time.
The image above which shows the states of all the nanopores indicates that approximately 70% of the nanopores were functional and actively sequencing. This tells us that I loaded enough cDNA into the device and that the pores were working as expected!
Come back next week, where I will discuss the results of my sequencing and its implications for my project. Thank you for reading!