10 October 2018, Wednesday. For last week’s lab HW, I had to make a folder with three of my samples (CO-01,02 and 03), 25 sequences of the COI gene for Actinopterygii and at least one COI gene for Chondrichthyes (outgroup). To find those sequences, I had to log into Geneious and click NCBI nucleotide section and then type in COI Actinopterygii or Chondrichtyes on the search bar. I put all 29 samples into one folder and made an nucleotide alignment sequence.
First thing that I did was to clean up my alignment to begin and end at the same point. I just clicked ‘Allow editing’ and select the bases and deleted them. Looking at the first 20 columns of my alignment, I found 12 polymorphic nucleotides. Next I downloaded JModelTest2 from Canvas. I had to download Java to open up the file jmodeltest2 because my computer was not able to open up the file. After that, I went back to Geneious and export my edited alignment in Phylip format (relaxed) by choosing Export, selected documents’, ‘Phylip’ file type and relaxed Phylip file at the end. And using JModelTest 2, I opened up the exported alignment. Next I chose ‘Analysis > Compute likelihood scores’ from the menu. My alignment took about 5 minutes for the program to look for the likelihood for each of the models to be the best fit. After the calculations were done, I looked up AIC and BIC values (red, top scoring model) and found out that the values were the same with HKY +G.
Back in Geneious, I clicked Tools, Plugins and install the Bayes plugin. Then selected my alignment, right click and chose Tree then MrBayes. Changed the substitution Model to HKY, Rate Variation to Gamma, Outgroup to shark, Chain length to 10,100, Subsample frequency to 99, Burn-in Length to 100 and left the heated chains at 4. I then run the analysis and didn’t took that long. Double-click on new ‘Posterior output’ to open up the result and click on the ‘parameter estimates tab’. I got really bad graph as expected and also got bad Trace as expected because the run time was very fast. This time I changed chain length to 100,000 and bur- in length to 10000. It took about 10 minutes and I got a pretty good graph.
This time, I went to Tools, Plugins and installed RAxML. This time, instead of choosing Bayesian, I chose ‘Rapid bootstrap with rapid hill climbing’. Once RAxML was done, I clicked ‘RAxML bootstrapping trees and right clicked to choose Tree and consensus tree builder. After that I chose ‘Create consensus tree’ and ‘Support threshold’ of 50%, and then ‘ok’. I had a decent tree and for my report I had to take two sequences out because the branch was too long. The other steps didn’t work so i had to stop at PHYML part.
10/3/18 Wednesday. Today, I used Geneious to look at my forward and reverse reads, however my reads weren’t successful so I had to used the given three forward and reverse reads which are CO-01-Albacore, CO-02-Big eye tuna and CO-03-Yellowtail.
I received “An Introduction to Geneious” handout with instructions on how to use Geneious and do the exercises. I first downloaded all the forward and reverse reads and drop the file into the folder that I named “Jinwoo Kim DNA barcoding for Molecular Ecology”. Next, I looked both forward and reverse reads to see how good they were and the HQ% was around 90s so they were very clean. Using CO-01, I tried looking at the sequence by clicking sequence view and also tried editing the nucleotide bases and etc… but never saved it. After that I copied the reverse sequences CO-01 into the forward reads folder. I then selected both of them and right clicked to choose ‘De novo assembly’ to assemble the two reads. Next, I clicked the created file named ‘assembly’ and zoom in to see the overlap of reverse and forward reads. Mine had high HQ% values so I was able to cut the ends of both sequences and was good to go. After the trimming the mess, I saved the file and choose ‘Generate consensus sequence’ and clicked ok. I BLAST the assembly ‘consensus sequence’ and resulted to have scientific name of the organism that has the highest matching sequence to mine. I searched up the scientific name to see if its the correct fish. At last, I copy and paste 5-7 alignment to my created folder named “Fish barcode test alignment’ and selected them all to click ‘Multiple align’ and get a new ‘nucleotide alignment file’. I followed this steps for CO-02 and 03.
- CO-01—– Matched, Albacore=T.alalunga, CO-02—–Matched, Big eye tuna=Thunnus albacores, CO-03—–Matched, Yellowtail=Seriola quin queradiata.
- CTTTTCTGATG (first 10) and total of 18.
26 Sept. 2018 Wednesday
Our last field trip was to Mt. Tamalpais and the weather was hot and cold in between each stops. We first stopped at where I believe it was Alpine Dam and we walked down the trail and stopped at one point where there was little river and had to go through little adventure (and I got hurt little bit). Few of us went down to the river and saw some plants between rocks and also saw bunch of ladybugs having a party inside a bush. Here are some pictures.
After some adventure, we drove across the bridge and went up the mountain and stopped at a place where plants were growing on wet grounds. Here are some pictures:
Professor has told us that this kind of plants need to be on wet ground all the time.
Next we drove down to the coast to see the purple-flowered lupine that was the same but different color from last field trip to Pescadaro state beach. As we drove to the coast, the view was so good that I had to take a picture of it.
Anyways, we dropped at one point where we observed purple-flowered lupine and it was in similar environment as the yellow colored lupine. There weren’t a lot of them, but we were able to find pea-shaped flowers, immature fruit and old fruit. We even found one on the cliff that was growing on top of the rock. It was windy and cold and there were lot of birds creating white rock. I was thinking that these species might have been spread out due to the wind and maybe the birds. Here are some pictures of the flower:
From this field trip, I was more amazed by the nature than the field trip from last week. It was amazing how we were able to find the same species but different color that is 2 hours apart from each other.
p.s Sad that this was our last field trip as a class 🙁