RAD Sequencing and Library Building: 
Process and Preparation 
 

What is RAD sequencing?

Restriction-site Associated DNA Sequencing – or RADSeq – is a fairly new approach to genotyping and genome sequencing, in which DNA fragments are created from the genome that can then be used to identify genetic variations.[1] These variations can tell us important information about population structure, association mapping, and evolutionary genetics. RADSeq can also be used to identify quantitative trait loci – or QTLs – which can tell us about genes causing phenotypic variation.[2] Thus, the information gained from RADSeq helps us to learn more about the populations of US East Coast bottlenose dolphins and their genes associated with traits such as adaptations to hypoxia.

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How do we prepare for RAD sequencing?

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A lab in Durham performs the sequencing for us, so our job is to send that lab samples containing dolphin DNA. For this process, we start with our dolphin biopsy samples. Each sample contains skin and blubber – for this process, we are only interested in the skin, which contains genetic material. We cut a small portion of the skin (about 25% of the sample) away and digest it overnight in a digestion solution. This process breaks down the sample to make the DNA more accessible. Once digested, we separate the solid remains of the sample from the liquid and transfer the liquid to a vacuum. During the vacuum process, DNA In the liquid binds to a small silica in our sample tube, and the liquid is drained from each sample. Following this, we break the bonds of the DNA to the silica by adding water – this water and the DNA suspended in it make up our final extraction sample. Because the sequencing lab requires a minimum amount of DNA to perform RADSeq, we lastly read the concentration of DNA in each sample, allowing us to calculate how much of each water-DNA sample should be sent to the lab. A brief overview of this preparation process can be seen in the infographic to the right.

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What do we do with sequencing data?

We are currently working on our third iteration of this process. Each time the lab performs RADSeq, a new RAD Library is created. Each library contains the resulting sequenced datasets, and we can then analyze the libraries for variations of certain genes, commonalities and differences between different populations, and more. We are in the initial stages of analyzing our results from our most recently created RAD libraries and are excited to see how these results add to our exisitng knowledge of these populations!

Sources: 

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[1] “RAD Sequencing,” Floragenex, https://www.floragenex.com/rad-seq, accessed January 28, 2022.

[2] Miles, Cecilia M. & Wayne, Marta. Quantitative Trait Locus (QTL) Analysis, Nature Education 1(1).