In this lab, we were able to download a program, Geneious, which allowed us to access the sequences produced by our DNA extractions of animal tissue from sushi. From the 4 samples of DNA, only 2, GS01 and GS02, were viable to produce sequences from both the forward and backward primers. Starting with GS01, the forward primer sequence had an HQ (High Quality) of 74.2% and the reverse primer sequence had an HQ of 25.7%.Using the DeNovo feature, we were able to combine the forward and reverse primer sequences and edit the sequence to produce a consensus sequence with an HQ of 81.9%. This number was produced only after editing sequences, which consisted of cutting off the ends of the sequence and erasing or fixing single bases. GS02 was subjected to the same DeNovo feature and editing process to produce a consensus sequence with an HQ of 78.8%.
We then were able to BLAST (Basic Local Assignment Search Tool) each of our consensus sequences, which allowed us to search a database for highly similar sequences. We were able to determine how well the sequences matched based on the Grade of the sequence. We selected 5 species to compare the consensus sequences to. Using this tool, I was able to conclude that the sample labeled GS01 was most likely Thunnus Albacares, which is a yellowfin tuna meaning the sushi market was in fact selling tuna. Sample GS02 was also analyzed using the BLAST tool and after comparing the consensus sequence it was determined to most likely be Seriola quinqueradiata, which is a yellowtail fish as advertised in the sushi market.
While the sequences were very similar, there were polymorphisms. For GS01, there were 4 polymorphisms which resulted from editing since some bases were erased due to convoluted fluorescence. There were also may bases that were not definitively determined, thus the were discrepancies in those as well. Base 511 had GS02 consensus sequence of a T nucleotide, however, there the rest of the species had a consensus in which there was no base there. There was also a similar situation at base 498. At base 121, the fluorescence was confusing and caused a polymorphism which is depicted in figure 2.
When GS02 was compared to 5 other sequences, there were 22 polymorphisms. Although there were many polymorphisms, this was mostly due to including a 2 species that were not as similar to the sequence that were included in the comparison. However, when GS02 was compared to just the Seriola quinqueradiata, there were no polymorphisms. An example can be seen in Figure 3.