This week in lab we used our fish DNA sample results to use Geneious and analyze the genomic data. None of my samples fostered adequate results (I think this was due to exposure to soy sauce and having to stay in my backpack for more hours than originally planned). Because of this issue, I used a sample that was sequenced prior, called NS01, NS02, and NS03.

NS01 was labeled as yellow tail, NS01 was halibut, and NS03 was bluefin tuna. To start out the analysis I was personally worried because I tend to struggle with computers and new software. I made myself slow down and follow each step of the handed out protocol and found it to be easier than expected. I was surprised with the ability of the site to perform the BLAST so quickly and utilize the NCBI data base within the same forum and at such a fast rate.

I faced a few bumps in the road outside of not being able to use my original data. I accidentally deleted my first sequence assembly as well as the folders with my reverse and forward transcripts, but luckily after having practiced with the software I was able to quickly re-download everything and start from scratch.

Results:

NS01 served as yellowtail

Top result from the BLAST:  Seriola quinqueradiata (Japanese amberjack-yellowtail) 

Polymorphisms

1. BP: 445 (A instead of G)

There was only one polymorphism after deleting all of the ambiguities (N) with a hit start and hit end in the high thousands. Therefore my first sample was served as the correct fish.

NS02 served as Halibut

Top result from the BLAST: Paralichthys californicus voucher – California halibut

Polymorphisms

The reverse of the genomic data NS02 carried greater than 20 polymorphisms throughout the entire comparison whereas the forward and all other vouchers had no polymorphisms despite a 100% match.

1. BP: 403 (T instead of C)

2.  BP: 413 (T instead of C)

3. BP: 418 (G instead of C)

4. BP: 421 (G instead of T)

5. BP: 428 (C instead of T)

6. BP: 435 (G instead of A)

7. BP: 438 (T instead of C)

8. BP: 442 (T instead of C)

9. BP: 443 (C instead of A)

10. BP: 458 (C instead of A)

NS03 served as Bluefin Tuna

Top result from the BLAST: Thunnus orientalis isolate – bluefin tuna

There were no polymorphisms present when comparing the matching sequences. With a 100% match and no polymorphisms it was surprising to see a slightly less than optimal hit start and end number below 1,000.

9/19/18

I was sick during this field trip so I was unable to attend. The class went down the coast (south) to find  Lupinus arboreus for collection. We will be extracting DNA from these plants to better understand their genetics and population patterns.

We took a lab field trip to see purple flowered lupine and Mimulus cardinals. We drove to Mt. Tamalpais followed by a trip to Stinson Beach and down the coast. Mount Tamalpais was supposed to have the red flowering Mimulus cardinals along wet water ways. Stinson Beach Coastal Cliffs are the environment of the purple flowered lupines.

Unfortunately, we were not able to find a flowering plant of Mimulus cardinals (we did find the plant, just not flowering). On the hike down to the water, it was noted that it would be extremely difficult for populations to share genomic information due to the sheer cliffs and hills. As the Mimulus cardinals is pollinated by humming birds, it is hard to imagine a humming bird being able to fly such long uphill distances between the different populations. This leads to geographic isolation that may seem small, but has a large effect on the Mimulus cardinals. We also discussed one of the oak tree family trees that was severely suffering from the fungal disease “sudden oak death.” The trees have very little genomic diversity leaving their population extremely susceptible to this disease. This is an issue with losing genetic diversity within a population because individuals do not have the possibility of diversity that may be needed to fight off pathogens.

On our search for the purple flowered lupine, we traveled closer to the coast near Stinson Beach. There were many plants in the area, but less flowers. As a self pollinating population, they appeared to be thriving even in the harsh winds of the coast line. The plant had almost soft looking leaves and a green color that was different from the other foliage present. In the areas of the lupine, many fennel plants were often present giving off their strong licorice smell. Mt. Tamalpais (looking for Mimulus cardinals)

Creek where populations of Mimulus cardinals would be found

Dr. Paul’s Secret Mimulus cardinals Plant along a small wet area

Drive from Mt. Tam to Coastal Area of the Purple Flowered Lupine

Environment of the Purple Flowered Lupine

The Purple Flowered Lupine Plant

Natalia Whitney

09/12/18

Following the lab where we amplified our DNA from the fish samples, we set up another gel electrophoresis to see the amplified gDNA. Using my 3 samples and loading dye we ran another PCR. The gel was provided by Dr. Paul so I did not have to make the gel. One of my table mates loaded the gel and checked to make sure the black was to black and the red was to red. We ran our gel, but unfortunately we only had 2 viable results for our entire table. Instead of performing the exosap clean up, we redid our gDNA collection. We followed the same protocol as last lab making sure to be careful with the master mix. I also vortexed my samples again before diluting them to make sure there were template strands available to be amplified. I placed my samples in the thermocycler prior to lecture section of lab. Hopefully the amplification and gel run will show properly amplified DNA for this second round.

Natalia Whitney

09/05/18

To get my fresh fish, I went to Balboa Teriyaki (3536 Balboa Street San Francisco) on 9/4/18. I went with my housemate and she bought a poke bowl that had tuna and salmon while I got yellow tail sashimi. I put the 3 samples of fish in the tubes and placed them in the freezer.

In lab, I followed the protocol for DNA extraction from animal tissue that was handed out in class. Yellow tail was labeled as NW01, Salmon NW02, Tuna NW03. I cut a small piece of the yellow tail and it weighed in at 3mg. I used that piece to visually judge the cut sizes of the tuna and salmon because there was a long line for the scale.

After the pieces were cut, I added the 100 microL of extraction solution and then tissue prep solution to 3 separate microcentrifuge tubes and labeled them accordingly. I made sure to mix the solution throughly, but gently as the protocol states. I then added each of the fish samples to their correctly labeled tubes. I mashed the samples up using a pipet tip, but it was difficult to get everything broken up. The tuna sample was especially hard for me to completely break up. After letting them sit at my desk for 10 minutes, I incubated them for 3 minutes on the heat block (using a timer on my phone to keep track). Once the 3 minutes passed, I added 100 microL of neutralizing solution to the tubes. I vortexed each tube for approximately 5 seconds to make sure everything was mixed properly to stop the reaction.

Following the collection of DNA from my samples we ran gel electrophoresis to confirm the presence of genomic DNA. The gel was already made and provided by Dr. Paul. Each of my lab mates pipetted the loading dye for their samples onto the parafilm. I wrote down the order of our samples in my notebook so we could keep track of our own samples. After adding each of our gDNA (3 microL) onto the 2 microL of loading dye, one of my lab mates pipetted all 5 microL into the gel. We double checked that red was to red and black was to black and then proceeded by placing the voltage at 145 volts. We then ran our gel for 15 minutes.

When we looked at our gel, all samples had gDNA except for 1 sample. We proceeded to perform the PCR steps for all of our samples. One of the lab mates wanted to make the master mix for our table so after diluting my gDNA (18 microL of water with 2 microL of DNA into a PCR tube), I added my DNA and master mix for each sample into fresh PCR tubes. I made sure to keep everything on ice to prevent degradation. I put my sample on the thermocycler to start the amplification process.

Balboa Terkiyaki

Balboa Terkiyaki
Yellow Tail

Balboa Terkiyaki
Tuna (red)
Salmon (orange)

Gel Electrophoresis Results showing gDNA (SquidSquad)

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