After discussing behavioral ecology in lecture we continued on with our wet lab. This week in lab I worked on setting up a qPCR from dilution (start) to placement into the heat block (finish). I had 5 DNA samples given to me to prepare dilutions for the class. After that everyone was given dilutions of 15 samples to be amplified via PCR. The DNA had to be diluted because PCR is sensitive to large amounts of DNA and therefore works better under lower concentrations. I assisted with making the master mix after placing 1 microliter of my DNA samples into individual PCR tubes. We decided to create enough master mix for 70 samples to ensure we did not run out of master mix between all of our table mates. The Taq was added last to prevent any immature reactions before mixing with the DNA samples. Something different about this PCR was the use of only one primer, not a forward and a reverse.

Analysis of EPIC Markers

After collecting the genetic sequences, we found out in the end that none of our genomes had amplified successfully during the PCR. Because of this we had to use prior collected data for our analysis of the EPIC markers. My EPIC marker was 5525 and I used 25 samples of Mimulus cardinalis and an out group TG248 (Mimulus lewisii).

I downloaded the sequences from Dr. Paul’s canvas under my assigned marker and imported them into Geneious. After assembling them using muscle and editing the sequences to line up, I identified an polymorphisms present. The columns that contained polymorphisms were 4-9, 10, 16, 21, 25, 27, 52, 66, 79, 82, 132, 181, 256, and 276. Using the polymorphisms and general scanning of different colored peaks, I identified any heterozygotes present. The columns I determined to have heterozygotes were 132, 108, 97, and 27. Two of these heterozygote columns were also polymorphisms (132 and 27). I recorded an polymorphisms with the IUPAC ambiguity codes.

After editing and recording my sequences, I continued on with the assembly of EPIC marker alignments. I individually assembled the forward and reverse reads and then built an alignment for each marker. After assembling, I created an alignment of all the assemblies, including my out group (which only had one sequence-not a forward and reverse). I renamed the consensus sequences to ensure the proper run of the Mrbayes tree.

The Bayesian Tree strongly indicated clades that were extremely closely related which may indicate a geographic relationship between these species.

Goal: After collecting the DNA from the Mimulus plants last week in lab, this week we had the goal of running the DNA using gel electrophoresis to confirm genomic DNA and if present then amplifying the DNA using PCR.

Methods: Before amplifying the DNA, we ran the samples on a gel to confirm presence of DNA. All samples were run and run fast to get a basic idea of the substance present. Once gels were finished and confirmed, we set up the PCR tubes with reagents needed. One of my table mates set up the master mix and we made sure all reagents were added in the correct order and kept on ice until used. The master mix was set up for 40 samples to ensure enough reagent was available to everyone at our table. Only 1 microliter of DNA was used and 19 microliters of master mix was used. This is because PCR works best with small amount of genomic information. The Taq was added last to the mastermix to avoid the start of the reaction before the DNA was added. Once all reagents and DNA was combined in the PCR tubes they were placed on the heat block for amplification.

Results: All of my DNA samples were adequately collected and showed up on the gel. Everyone at my table had genomic DNA present. Data on proper amplification of genomic DNA was shown on Monday (10/29). My samples as well as everyone at my table had genomic DNA post amplification showing that there was not a high amount of degradation.

Images:

Mine were the first wells

Image 1: Squid Squad DNA after PCR            Image 2: Squid Squad Genomic DNA

This lab focused on extracting DNA from plants from different locations all falling under the species of Mimulus cardinals or Mimulus guttatus. Only one of my three sample IDs were labeled with location and name which was JP1321 (A) Mimulus guttatus from Mill Creek Mono North. The other two samples had the IDs JP1297 (B) and JP1300 (C). The tissue samples from these plants were left in silica beads to soak up any moisture to improve the DNA recovery from the sample. Due to the difficulty of extracting DNA from plants caused by the cellular structure and cell wall, I used 3 small metal balls for each sample to break down the tissue into a powdery poultice. The epi tubes that contained the samples and the 3 balls were then shaken extremely fast with a modified power tool for 40 seconds. Carter volunteered to use the power tool to break up my samples. After the samples were in a powder I used the protocol handed out for DNA extraction. This included adding a heated reagent followed by 10 minutes in the water bath during which the samples had to be inverted every 3 minutes to ensure proper exposure. I then used the centrifuge to spin down my samples to get rid of any dry solids and/or dust. Following this, acetate was supposed to be added. I had a small issue here because I was handed an uncapped falcon tube and was told it was the acetate, only to find out after that it was truly ethanol. I had to restart the protocol from the beginning, but was luckily able to catch up with the rest of the class during lecture. After reaching the acetate step once again I spun down my samples for 20 minutes. I then added the supernatant with another reagent at a 1:1.5 ratio. My samples all had 400 microliters of supernatant so I used 600 microliters of reagent. This was then pipetted in a two step process (due to large volume size) into individual column tubes to start the DNA collection process. After the all the supernatant had run through the column assigned to that sample, the columns were then washed with ethanol. Once the ethanol washes were completed, it was time to get the DNA from the column into the fresh correctly labeled epi tube. To do this I washed with warm water, let it sit for 2 minutes and followed that by centrifuging the columns with the collection epi tubes. The result was collected DNA in epi tubes that will be amplified and further analyzed for proper collection.

This week in lab after going over our lecture, we continued with our fish DNA samples to evaluate Phylogenetic Inferences of our data. I used the handed out protocol to par because doing so really helped me move through the software the week prior. Before coming to class I had already picked out around 25 different COI genes from a multitude of different Actinopterygii species. I also picked out 1 organism from the class chondrichthyes as an outgroup. I used Neotrygon kuhlii which is the bluespotted stingray. I had originally had picked another organism for this, but after aligning the sequences I realized that the genomes did not line up. Luckily, I was able to find the COI gene for the stingray and it lined up perfectly with my other sequences.

It took me awhile to troubleshoot finding the proper organisms through the NCBI nucleotide section in Genius because a bunch of random organisms would show up and if I got too specific in my search I got academic articles, not sequences.

Once my sequences were lined up I realized there were many polymorphic columns, at least in the first 20 columns. There were a total of 15 polymorphic columns in the first 20, but not all sets of 20 had this many. Others had anywhere from 5-1o columns that were polymorphic (if counting in sets of 20).

On the jModelTest my best model for AIC was JC 10975.525 . For my BIC it was JC 11182.16.

Initially I had a hard time figuring out how to get the plugins for the different tree models, but thanks to Dr. Paul I was able to figure it out:)

My maximum likelihood clades with the highest support also match my Bayesian analysis.

Issues: My folders kept being deleted so I had to redo this multiple times.

Organisms used for comparison:

Aligned Sequences:

AIC:

BIC:

Parameter estimates:

Trace:

Tree:

Maximum Likelihood/Consensus Tree:

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)