Library Preps before Sequencing our corals

Dear backers, we have been for some period in silence through thischannel, but not at all stopped in our project!... During these months we havebeen collecting the last batch of totally recovered coral colonies in May, sosix months after the climax of the bleaching event. In the Lab, we havefinalized all the DNA extractions of the available samples, and have organizedall those DNA extractions in little tubes and plates.

 

Now from those almost ~600 coral samples! We are proceeding to thenext step: The Library Preps. This term is a bit confusing for manypeople, as there are several concepts referred to as “Library Preps”. In ourcase, this has nothing to do with books, but is a way to organize your samplesby codes, as the librarians do in a library. This is essential to know whichsequences belong to such or such sample, out of the millions of them that willcome out from a sequencer. Without the special unique barcodes added to each ofyour samples, analyzing the data would be quite impossible.

 

The first step on a Library Prep is to amplify the target region ofyour DNA samples by millions of copies, and this target region is called ingenetic language “amplicon” (from amplification). In this way at the end, you basicallyend up with only that region to focus out of all the long, complicated metagenome thatcomposes a coral holobiont. As we mentioned in the project resume, in our case,the 2 target regions are the ITS-2 for zooxanthellae/microalgae (Symbiodinium) identification, and the16S region for determination of the associated bacterial communities. You canobtain millions of new copies of your target amplicon by PCR (Polymerase ChainReaction). This reaction is done in a ‘Thermocycler’ that is like a very potentoven that can change wide ranges of temperatures very very fast (inmilliseconds), and perform very quick and precise cycles of temperatures andtimes.

 

A normal example of a cycle of a PCR has a very high temperaturestep that separates the 2 DNA strands, the ‘Denaturation step’, then the‘Annealing step’ of lower temperature when the ‘Polymerase’ (the enzyme thatbuilds the DNA) and ‘Primers’ (short pieces of known DNA that are complementaryto the beginning and end of you target region) can attach to the DNA, and the‘Extension step’, when the new copies of DNA amplicons get synthesizedaccording to the template by adding new nucleotides (A,C,G,T).

 

These cycles repeat several times and produce in each cycle copies of your target region. At the end of the whole PCR reaction you end up with millions of copies of your amplicon.

 

A PCR reaction has a very specific mixture of reagents that you add along with your DNA template. These are normally composed of:

- DNA template

- Polymerase (enzyme that adds nucleotides complementary to your template)

- Nucleotides (the building blocks used by the polymerase to make copies)

- Primers Forward and Reverse (short pieces of DNA complementary to the beginning and end of the target region that mark the polymerase where to start and where to stop synthesizing DNA)

- Buffer (a liquid that provides the required ‘ambient’ for the reaction to occur)

- Electrolites (also provide the correct atmosphere for the reaction.

This ‘magic’ cocktail will allow the PCR to happen, and once they are well mixed, the reaction takes place in the ThermoCycler.

 

 

The way Next Generation Sequencing on Illumina platform works isthat the amplicons get barcoded automatically during the amplification in thePCR. This is because the specific Primers we use in the PCR already contain abarcode in their sequence that will remain in the DNA amplicon copies after thePCR. By combining a Forward and a Reverse primers with 2 barcodes (what iscalled ‘Dual Indexed’), you obtain unique coding for each of your samples.And that specific code will be used to identify the sequences coming out fromthe sequencer.

 

After you PCRs, you can check if the reaction worked out well, bycharging your PCR product in onto agarose gels with a dye that specificallyallows visualize DNA. Here is an example of the first PCR done on a first batchof 96 coral samples. Each band corresponds to the amplification of the ITS-2region for microalgal sequencing and analysis.

 

Well, pretty intense this Lab Note!... We hope your eyes did not fall over, or your brains did not get too warm reading this… But we wanted to show you and make you understand a bit what we are doing now. Hopefully you got to learn a little of genetics too, and that is a pleasure for us.

We look forward to give you further updates with more cool data!

Cheers!