This lab period utilized Geneious, a program that performs various functions on DNA and protein sequence data, to analyze the data collected in Lab 02. Both the forward and reverse of EVR03 succeeded while EVR01, EVR02, and EVR04 did not return any usable data. To substitute, I am using the extra code BP03 which was reported as albacore tuna.
Forward and reverse primers of EVR03 and BP03 were obtained and put through Geneious and were assembled in their respective samples. A new folder was made and the documents were copied and de novo assembled. The sequences were trimmed at the ends where confidence was low and some corrections were made to ambiguous codes where there was a discrepancy between the base calls of forward and reverse primers. Consensus sequences were generated and the sequences were BLASTed and the top hits were compared against what the fish was advertised as. An alignment was built of the sequences with some of the hits–EVR03 was tested against 8 different hits while BP03 was tested against 9 different hits. The alignments were built with Multiple align and muscle alignment with default settings.
- For EVR03, it was listed as mackerel–the BLAST revealed that the sequence was in the Scombridae family and had a grade of 99.2% for both Thunneus obseus and Thunneus albacares, though I am inclined to believe it is T. obseus because it’s slightly higher than T. albacares in the BLAST details. For BP03, it was listed as albacore tuna. The BLAST revealed the sequence was closest to Thunnus alalunga, which is, in fact, the albacore tuna. This result had a grade of 99.7%.
- Polymorphisms in EVR03’s nucleotide alignments were found at sites 178 and 292. Ambiguous codes weren’t found in sites with polymorphisms. The first ten polymorphisms in BP03’s nucleotide alignments were found at sites 19, 31, 46, 277, 283, 301, 328, 352, 373, and 415. There were 18 polymorphic sites in all.