The colorful world of sequencing!

In this post for our Online Science Exhibition, PhD student Igor Baars from The Hogberg lab at KI exhibits the colorful world of sequencing.

Hej everyone! Igor here again, this time to show you a little around for the exciting world of sequencing.

When working with cells, you often must work sterile, in which case you will work and do all experiments in a laminar flow hood. Not all samples have to be sterile in culture though. For example, when you are working with C. elegans (an amazing tiny little worm), you often already culture them with bacteria (as that is their food), so in those cases you cannot really work fully sterile.

When growing cells, we must often work sterile and in a separate hood.

Another kind of model organism to use is the C. elegans, a fascinating tiny worm. Say you are done with your experiment and to know how it affected gene expression. A good way to see that is by analyzing the messenger RNA (mRNA) of each gene. Now to get to this information, you need to isolate these molecules first, a procedure that is sensitive to contamination from the outside. For this reason, we can also choose to work sterile in a flow hood to reduce contamination or possible degradation of the mRNA.

A hood for sensitive RNA work.

Once you have your mRNA sample, it is time to turn your sample into a sample ready for sequencing, otherwise known as a sequencing library. Usually you do this by first turing your RNA into DNA and add some extra sequences or proteins that allows them to interact with your sequencer. Once that is done, you must see if your library is good to go. Often you can do this on a Bioanalyzer, in which you can see the length distribution of your sample. The idea is to get three peaks: a 35 and 10380 peak from the machine itself and then a single peak from your sample. However, if your preparation fails that peak can turn into several peaks, a large smeary peak or something in the middle.

A bioanalyzer can show you what is in your sample.
But sometimes it goes wrong…

Once your library is ready to go, you can start sequencing. Often, it is straightforward, just add your sample to the chip, press a few buttons on the machine and you are done! Your Illumina sequencer turns from a nice happy green to a serious blue and goes to work.

Our little illumnia NextSeq, all happy and error-free!
Our little NextSeq when it is doing something (in this case a routine wash).

Once your sequencing run is done and the machine goes back to happy green, you will see a small plot saying how your run went and an indication of the quality of your reads. If it looks like your reads passed the quality filter, you can get your results and start analyzing them!

Standard output screen of a run shows an indication of the quality of the run

Now that is all I have to say for now, hope you enjoyed this tiny tour of sequencing! Stay tuned for more exciting stuff for our science exhibition from my other network fellows.

See you in the next blog!

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