The Truth Revealed

On Wednesday, September 27th, we used Geneious to analyze our PCR sequences and finally elucidate the identities of our fish samples.

Procedure:

  1. We received our DNA sequences from the PCR procedure in previous weeks.
  2. Next, we downloaded the sequences from Canvas and imported them into Geneious, a sequence alignment, assembly and analysis software. I imported CMN1, CMN3, and JRP01. The first two came from my fish samples and the third came from one of Prof. Paul’s.  My other sequences, CMN2 and CMN4, did not contain enough clear data to be used in Geneious analysis.
  3. We followed numerous steps of a tutorial that Prof. Paul provided us with. This tutorial helped us understand how to recognize high quality scores and forward and reverse reads, as well as basic characteristics of the sequences.
  4. Next, I assembled the forward and reverse sequences together for each sequence (CMN1, CMN3, and JRP01). We deleted unreadable bases and manually edited ambiguities.
  5. For each sequence, I generated a consensus sequence. and used BLAST to search the BLAST server at NCBI for highly similar sequences. The matches with the top bit-scores and E value of 0 helped me determine which species had the closest sequence similarity.
  6. I used Google to search for the scientific names of the species contained in the matches with the highest top-bit species in each Hit table.
  7. I found that CMN1 was either Pacific or Atlantic bluefin tuna, CMN3 was Atlantic mackerel, and JRP01 was Chinook salmon.
  8. Next, I created new folders to create three different fish barcode test alignments. I pasted the top 8 BLAST results from each fish sample into its respective folder.
  9. I created three multiple alignments, and analyzed them to determine how many polymorphisms were present in each.

Exercises:

  1. The first sample (CMN1) matched the fish I was told to have been served. However, my third sample did not match, as I was told that I received yellowtail. As both mackerel and yellowtail were on my plate, I may have made an error in labeling my samples at some point in the procedure.

CMN1–BLAST=either Pacific or Atlantic bluefin tuna

CMN2–data not available

CMN3–BLAST=Atlantic mackerel

CMN4–data not available

I also analyzed JRP01. After I used BLAST, I found that this species was Chinook salmon (also known as king salmon).

2. CMN1 alignment: 1 polymorphism

CMN3 alignment: 0 polymorphisms

JRP01 alignment: 0 polymorphisms

3. I built an alignment using the consensus sequences from each of my samples, as well as sequences from the CO1 gene from 31 Actinopterygii species and one ray species (Torpedo torpedo). 

 

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