On October 3rd we were introduced to the Geneious software program. The lab revolved around the interpretation of the sushi sequence we collected the durning the last laboratory session. Since we had already downloaded the program onto out laptops before the class, we were able to begin the lab right away. The lab began with an introduction allowing us to get familiar with the program. First, we created a new folder under ‘Local’ and named it ‘Fish barcodes.’Next, we downloaded the DNA barcoding sequences from Canvas which contained both the forward and reverse sequences. The files was imported into ‘Fish barcodes.’ Following this step, we double clicked on one of our individual forward files for our fish DNA. For example, I selected my LMC1 file which showed clean peaks that were evenly spaced towards the center but fluctuated on the ends, low quality. Two of my fish samples did not run. My files LMC3, LMC4 and LMC5 failed completely.
Soon after we continued the lab following the protocol which directed us to further explore the system in the pop-up of the selected file. We clicked on ‘Sequence’ view and ‘Chromatogram’ to see the presentation of base letters. The peaks were also analyzed to notice the height of the peaks and their relation to better quality. It was also pointed out that if something is low quality an ‘N’ will be presented. The quality score was observed by selecting the ‘Text View’ tab. The next tab observed was the “Info’ tab which allowed us to view information on the sequence, a feature that will be very useful in the future. The final tabs selected included: “Sequence view,’ ‘R.C.,’ ‘Colors,’ ‘Translate,’ ‘Base call quality,’ and the magnifying glass. All the features selected allowed us to familiarize ourselves with the features available for our use. The final step of this section directed us to make a change to the sequence and exiting out of the pop- up to see the ‘Save’ button that appears when someone attempts to exit a pop-up that had experienced changes.
After this portion of the protocol, the instructions led us to set up a forward and reverse sequence reads of the same sample selected. In this first case files LMC1 forward and reverse, Tuna sequenced DNA, were selected. LMC1 reverse file was copied and pasted into the “Forward reads’ folder. Once both reads were in the same folder they were selected and “De novo assembly’ was clicked. When the first pop-up appeared the settings were left the same and the ‘okay’ button was clicked this generated a new file with the name of the first file with the word ‘assembly included. The file was opened and magnified with the zoom feature. The two sequences aligned was depicted along with the consensus sequence. Next, the two sequences, forward and reverse, were edited on either end to delete base pairs that were not readable. The trimming process was saved by closing and selecting the save button.
Following the assembly process the generated file was selected and a ‘consensus sequence’ was generated with the default setting set. A ‘consensus sequence’ was created. Then by clicking the file and the ‘Blast’ feature, with default settings, a selection of 100 highly similar sequences was generated as a ‘Hit table.’ A new tab was opened for the top hit and the name of the similar fish sequence was googled to determine if the tuna I had sequenced was actually tune based on the similar sequence that was found.
The final step in the protocol required us to build an alignment with top 5-10 hits. We selected five different species and moved them to a new folder named ‘Fish barcode test alignment.’ The final file that was copied and pasted into the new folder created was the ‘assembly consensus sequence’ file for LMC1. Once all files were in the new folder and selected, “Multiple align’ was chosen. This step generated a new window in which ‘Muscle alignment’ was selected and default settings were left unchanged. The last file created was ‘nucleotide alignment file’ which present an alignment of all the sequences selected. The magnifying glass was selected to observe possible polymorphisms.
The process of BLASTing through alignment for the forward and reverse sequence was repeated for files LMC2 and NS02. There were slight differences when the steps were run again for the new sequences. The sample that was used to generate the DNA sequence for file LMC2 was done twice. As a result I worked with 2 forward and 2 reverse sequences instead of 1 forward and one reverse sequence. Aside from the slight difference all other steps were followed. The final file, NS02, did not contain the DNA sequence to my samples of fish. I used the data in the NS02 file because three of my samples did not run. They failed completely. Although not my data, the same procedure was completed.
- The sequence labeled LMC1 did present the species it was supposed to be which was tuna. The sequence LMC2 was supposed to present Escolar but actually presented Atlantic mackerel. This could be either because the restaurant did not provide the right fish or because I miss labeled the original sample tube. The NS2 sequence presented Paralichthys aetuarius, Cortez flounder when it should have sequenced Halibut.
- The first 10 polymorphic sites found: Number ,Base Pair letter,Color
- 7,A,Greenbrown/11,A,Greenbrown/16,T, Greenbrown/19, A, Greenbrown/22,C,Greenbrown/34,T,Red/46,C,Greenbrown/298,C,Greenbrown/ 301,C,Green/316,C,Greenbrown
- Total Polymorphic Sites: 15
- 5,6,7,10,12,18= less 30% identity 40,C,Greenbrown/243,C,Greenbrown/261,G,Greenbrown/654,A,Greenbrown
- Total Polymorphic sites: 16
- Total Polymorphic Sites: 50
This information was extracted from my original Geneious data collected. Due to a malfunction of Geneious I had to run the protocol again to obtain the new information I have now.