Lab Entry 6 – Geneious Gene Sequencing and Analysis
With the completion of DNA replication and running multiple PCRs, results of gene sequences were received in which analysis could take place. A program, Geneious, allows for this analysis by comparing the forward and reverse sequences of data found for each of the samples. All three of my samples produced sequences with high HQ% values. With all six of the forward and reverse sequences placed into a file on Geneious, a comparison could be made for each of the three samples.
First, the two matching sequences were selected and then ran through De Novo Assembly to piece the sequences together to match each other as accurately as possible. The program runs the forward and reverse sequences in the same direction for a comparison to occur. Any base pairs that were unable to be sequenced properly were deleted while some base changes were altered in order to match the other sequence based on the level of accuracy produced by the program. Once the sequences were trimmed and corrected to match each other, the sequence was compared to other sequences withing the Geneious in order to determine the origin of the sample. The program lists a multitude of potential matching sequences, providing a percentage of how closely they relate to each other. The sequence with the highest sample can be assumed to have the same DNA as the sample tested resulting in the genetic identification of the species of sample.
Exercises:
1). All of the samples were correct when comparing sequence identification to what the samples were marketed as in the restaurant. Chinook salmon, yellowfin tuna, and yellowtail were all correctly identified from the sequencing performed.
2). The salmon sample had no polymorphic sites when comparing my sequences to the sequences of the best matching species. The tuna sample had quite a few polymorphic sites where there were different base pairs at the same location. The yellowtail sample had two polymorphic sites near the end of the sequences but matched perfectly other than those two differences.
3). For the third part, 27 sequences were collected to go along with the three sequences from my samples for a total of 30 sequences to compare with. The sequences consisted of ray-finned fish species (Actinopterygii) and a few shark and ray species (Chondrichthyes).
