We conducted a double digest restriction associated DNA study on M. Guttatus. The first step was collecting samples which we did on two separate field trips that can be read about here:
- https://usfblogs.usfca.edu/rgutierrezvallejo1/
Next we extracted DNA from samples we collected as well as samples previously collected by our boy Alec. The extraction process is summarized below:
- https://usfblogs.usfca.edu/rgutierrezvallejo1/
Next we double digested our DNA using two restriction enzymes, that cut up the genome into many pieces, following the protocol found below:
- https://usfblogs.usfca.edu/rgutierrezvallejo1/
Following that we ligated unique DNA barcodes onto each of our individuals. Then we used PCR for two purposes: (1) to add a second table-based unique barcode and (2) to test if our library construction was successful. Our PCR was successful as evidence by photos taken by Prof. Paul uploaded to Canvas. After the test PCR, we did a larger reaction at 25µL that was identical, which will be used for the following steps. This was the last step we were able to do as a class. In a perfect world we will do the following steps. The next step would be size selection, which selects DNA of specific sizes. Specifically, we would target approximately 400-600bp fragments. Size selection can be done in three ways: (1) using an automated system called Pippen Prep of which we have one housed in the Suni Lab, (2) using gel extraction, or (3) use magnetic beads to isolate DNA fragments. After size selection, we would then normalize DNA samples, this means bringing all our DNA samples to approximately the same concentration. Having equal concentrations makes it more likely that more DNA fragments would be sequenced. The final step would be to combine all size selected normalized PCR products into one vessel and then we would run these samples on any Ilumina sequencer (aka Wall-E).
Sequencing would take approximately 16 hours and if successful would generate 10s of millions of reads. These data would be run through a bioinformatics pipeline (Help Dr. Z!). Ultimately we would align these sequenced data with the published M. Gattatus genome and call SNPs. Finally we would use these SNPs to infer population differentiation using a metric like Fst and assess population genetic diversity looking at things like the number of alleles, allelic diversity, etc. Based off what I know about M. Gattatus I would expect populations that are geographically divergent to be genetically divergent as well due to local adaptations to environmental conditions.
