- First, I labeled 3 2.0 mL tubes with my sample codes (KRS1-3 respectively).
- I added 3 sterile 3.2-mm stainless steel beads to each tube.
- Then, I added a small amount of leaf tissue into each tube, cleaning the tweezers between tubes to avoid contamination.
- Professor Paul loaded the tubes within a tube rack into the modified reciprocating saw rack and mounted the rack to the saw. He then turned it on speed 3 for 40 seconds.
- After, I centrifuged the tubes for 15-20 seconds at a fast speed to bring the plant dust down.
- I added 440 mL of preheated grind buffer to each tube.
- I incubated the buffered grandame at 65 degree C for 10 minutes in a water bath, mixing the tubes by inversion every 3 min.
- I then added 130 mL 3M pH 4.7 potassium acetate, inverted the tubes several times, and incubated the tubes on ice for 5 min.
- I centrifuged the tubes at maximum force for 20 min.
- I labeled new 1.5 mL tubes with the sample IDs and transferred the supernatant to the sterile tubes, avoiding transferring precipitate.
- Then, I added ~600mL of binding buffer to each tube, inverting to mix the solution.
- I added 650 mL of the new mixture to Epoch spin column tubes and centrifuged for 10 min, discarding the flow-through in a beaker.
- I repeated step 12 with the remaining solution.
- I washed the DNA bound to the silica membrane by adding 500 mL of 70% EtOH to the column and centrifuging at 15,000 rpm for 8 min until all liquid has passed through the membrane. I discarded the flow-through.
- I repeated step 14.
- Then, I centrifuged the empty columns at 15,000 rpm for an additional 5 min to remove any residual ethanol.
- I discarded the collection tubes and placed the columns in sterile, labeled 1.5 mL microcentrifuge tubes.
- Finally, I added 100 mL preheated pure sterile water to each tube and let it stand for 5 min before centrifuging for 2 minutes at 15,000 rpm. The solution left in the tube is the DNA.
I created an alignment of 25 COI sequences from Actinopterygii and my fish DNA barcode sequences, including one Chondrichthyes sequence as an out-group. I did this by searching COI and Actinopterygii/Chondrichthyes in the NCBI nucleotide program in Geneious. I chose sequences of similar lengths to my fish DNA sequences. I edited the alignment by choosing ‘Allow editing’ so that the sequences began and ended at the same point. When looking at the first 20 columns of my alignment, there were nine polymorphisms.
Then, I downloaded jModelTest2 to determine the best model of molecular evolution for my sequences. In Geneious, I exported the alignment in Philip format (relaxed). Then, in jModelTest2, I opened the file by clicking ‘File’ and ‘Load DNA Alignment’. Then I clicked ‘Analysis ‘and ‘Compute likelihood scores’, keeping the default settings. I looked at two methods, Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC). I did this by clicking ‘Do AIC/BIC’ respectively under the analysis window, keeping the default settings, and clicking ‘Do AIC/BIC calculations’. The best model based on AIC was JC, which was the same as the best model based on BIC.
Next, using Bayesian inference in Geneious, I selected my alignment, right clicked and chose ‘Tree’, then ‘MrBayes’. I used JC69 for the ‘substitution model’ and equal for ‘rate variation’. I kept the ‘gamma categories’ selection at 4. The ‘outgroup’ was put on HM422916, the sandshark outgroup I found earlier. For the initial ‘chain length’, I set it to 110,00. The ‘burn-in length’ was 10,000. I used the default setting for all other parameters. After running this analysis, I opened the ‘posterior output’ line and clicked on the ‘parameter estimates’ and ‘trace’ tabs.
Next, using maximum likelihood to infer a phylogenetic tree, I installed RAxML. I chose a similar evolutionary model to the MrBayes model and chose ‘rapid bootstrap with rapid hill climbing’. Once RAxML was done, I right-clicked the new line and chose ‘Tree’ and then ‘Consensus Tree Builder’. Then, I clicked ‘create consensus tree’ and ‘support threshold’ of 50%. The clades in the resulting tree did not match the ones from the Bayesian analysis.
Using Geneious, we were able to match our fish DNA to DNA in the database. To do this we first copied the reverse sequence from the ‘Fish barcode Reverse Reads’ folder and pasted it into the ‘Forward Reads’ folder. Then, we selected the forward and reverse sequences of the same ID and clicked the Align/Assemble tab, choosing ‘De novo assembly”. Using the default settings, a new file appeared containing the consensus sequence for that fish DNA. We edited the sequence to clean up any discrepancies and saved these changes. We then right-clicked this file and chose ‘Generate consensus sequence’. We used this file to BLAST our sequence by right-clicking the file and selecting ‘BLAST’, keeping the default settings. Another file appeared, allowing us to see a set of top matches for our sequence. These matches told us what species our fish DNA most closely resembles. We created a new folder for a fish barcode test alignment and selected the assembly consensus sequence and 5-10 hits from the BLAST search, pasting these into the new folder. We then selected all of these sequences in the new folder and right-clicked, choosing ‘Multiple Align’ and then ‘Muscle Alignment’ with default settings. A new file was generated, showing polymorphisms between our DNA and other DNA of the same species.
Unfortunately, only two of my DNA barcodes were successful: KRS1 and KRS4.
|Number||Unique ID code||Restaurant Species Name||DNA Barcode Species Name|
|1||KRS1||Tuna||Thunnus Albacares (Yellowfin Tuna)|
|4||KRS4||Salmon||Salmon Salar (Atlantic Salmon)|
The successful DNA barcodes matched with the species specified at the restaurant.
For KRS1, there were 11 polymorphic sites found in columns 31, 167, 228, 324, 332, 333, 344, 346, 371, and 387.
For KRS4, there were 10 polymorphic sites found in columns 69, 586, 590, 611, 612, 614, 625, 628, 631, and 632.
On Tuesday, September 24, 2019, we traveled back to Mount Tamalpais, this time stopping near Muir Beach to collect and view samples of mimulus guttatus. We stopped at a natural spring to drink the water and observe the flowering mimulus guttatus. We then traveled to a shaded creek bed to find more mimulus guttatus samples. Alec collected these samples to include in our lab.