October 3, 2018
The introduction to Geneious lab segment illustrated the methods of utilizing the Geneious program. After completing the installation process, several sequences from the fish samples amplified in the previous labs were tested to identify whether the labels from the restaurant matched the information presented by the genomic data. These were the steps followed to complete the exercises using Geneious:
- Retrieved forward and reverse reads from Canvas and installed the files into Geneious.
- Copied and pasted a corresponding reverse read into the same folder as the forward read (eg. YH01_fwd, and YH01_rvs in same place).
- Assembled both forward and reverse reads onto same document using “De novo assemble”. Document was then named “YH#_ASSEMBLY”.
- Deleted any unreadable end bases.
- Modified bases that were illegible by comparing forward and reverse reads (eg. deleted unreadable bases on reverse strand and replaced it with legible base shown at homologous nucleotide site on forward read).
- Saved modified version of consensus sequence.
- Generated consensus sequence, which created new document under name “YH#_ASSEMBLY consensus”.
- Used BLAST on consensus sequence to search NCBI database for similar sequences.
- New folder of sequence matches appeared on menu.
- Identified whether sequenced sample matched labels provided at restaurant.
To build an alignment, these steps were taken:
- A new folder, named Fish Barcode Test Alignment, was created to host the barcode alignment documents.
- YH01_ASSEMBLY consensus sequence was copied and pasted into the newly created folder.
- 5 BLAST results within the top 100 hits for YH01 consensus sequence were selected and copied.
- The selected matching sequences were pasted into the Fish Barcode Test Alignment folder.
- All documents inside of the new folder were selected and the ‘multiple align’ function was used.
- A new document consisting of the nucleotide alignment of the five selected results and YH01 consensus sequence was generated.
- Polymorphisms were identified.
Due to unfortunate circumstances, the only samples sequenced using Geneious were YH01, YH02, and ARA01 (named YH_ARA01). YH01 was the sample of Tuna as depicted by the figure above, whereas YH02 consisted of a sample of Escolar. YH_ARA01 was an assigned sample of Yellowfin Tuna, as a replacement for the other samples that were unable to be properly amplified. YH01 and YH_ARA01 matched the species being served, though YH01 was not labeled as specifically. However, both samples were sequence-referenced through BLAST and the highest match percentages indicated that the species was Thunnus albacares, which was Yellowfin Tuna. On the other hand, YH02, the sample of Escolar, revealed a mixture of Thunnus albacares, and two different bacteria species. YH02 was sequenced three times, and broken into consensus sequences of YH02A, YH02B, and YH02C. BLAST results for YH02A matched the sample with Thunnus albacares, despite the differences in the sample’s appearance (Escolar versus Tuna), which indicated a degree of contamination between samples YHo1 and YH02. In addition, sequence YH02B matched most closely – though the percentage was fairly low – to Pesudomonas ludensis, a bacterial species that causes spoiling of milk, cheese, meat, and fish. Similarly, YH02C yielded a high match percentage with Pesudomonas fragi, a species of bacteria generally responsible for daily spoilages. YH02B and YH02C showed that not only was there contamination between samples, but also preservation issues regarding the sample of Escolar obtained.
According to the alignment built with YH01_ASSEMBLY consensus sequence, there were approximately 16 polymorphic sites (nt 681, 666, 426, 417, 375, 303, 288, 285, 279, 33, 25, 21, 12, 9, 6, 3) among the five chosen BLAST results. The first ten polymorphic sites were at positions: 3, 6, 9, 12, 21, 25, 33, 279, 285, and 288.