Rationale:

The purpose of the lab was to review the annotations and good poster design ideas.

Tools: 

• DNA Master 
• GeneMark  
• NCBI Blast 
• PhagesDB Blast 
• Phamarater 
• HHPred 
• Phage Notes 

Procedure: 

1. Gene Annotations for NapoleonB were reviewed.  
2. Gaps in the Open Reading Frames were discussed and reviewed.  
3. Changes were made to Gene 96 of Napoleon B.  
4. New annotation was updated to PhageNotes.  
5. Important aspects of a scientific report were reviewed.  

Results: 

Annotation of Gene 96:  

Conclusion: 

Gene 96 was annotated again since a better value and hit was found. The start value of gene 14 was inputted incorrectly, so edits were made.  

Future Work:  

Groups will be assigned for the poster project and designs for the poster will be started.  

Rationale:

The purpose of the lab was to revise NapoleonB’s genes, and to create a final abstract.  

Tools:  

• DNA Master 
• GeneMark  
• NCBI Blast 
• PhagesDB Blast 
• Phamerater 

Procedure: 

1. Groups were assigned for annotations and abstract.  
2. NapoleonB’s Gene 96 Annotation was re-evaluated because of a large gap between gene 95 and 96.  
3. Coding potential between gene 95 and 96 was viewed and evaluated.  
4. A place which appeared to have a high coding potential on GeneMark was added to DNA Master as a test gene.  
5. Results were collected with the product of the test gene on PhagesDB and NCBI Blast.  
6. Phamerater was used to compare different AM cluster phages.  

Results: 

The results on PhagesDB and NCBI Blast displayed that there were no hits for the particular coding region, which was a reverse ORF. Therefore, the annotation of Gene 96 was not changed.  

Conclusion:

More knowledge was acquired on DNA Master about how to test gaps and see if there was a hit. Even though GeneMark displayed a high coding potential on the graph, the results displayed no hits.  

Future Work:

Next class we will re-evaluate the genes and recheck the annotations of the genes.  

Rationale:

The purpose of the lab was to annotate the remainder of NapoleonB’s genes with the knowledge and skills acquired in the past few weeks.  

Tools: 

• DNA Master 
• PhagesDB  
• NCBI BLAST 
• Phamerator 
• HHPred 
• GeneMark  
• PhageNotes 

Procedure: 

1.  FASTA file of NapoleonB was opened on DNA Master.  
2. Genes were located and protein sequences were used in PhagesDB, NCBI BLAST and HHPRED.  
3. Phamerator, and GeneMark were used for Synteny and  Coding Potential. 
4. Annotations of gene 14,15,16 and 96 were completed and saved on PhageNotes.

Results: 

Annotations of Gene 13,14,15,16, and 96 

ORF’s for genes:

Conclusion: 

Gene 13, and 15 had no function, but genes 14,16,96 had a function.  Gene 14 and 16 were major tail proteins supported by NCBI BLAST, PhagesDB, HHPred, and Phamerator.  It was difficult to determine the start of gene 96 since there was a huge gap between gene 96 and 95. Also there was a slightly large overlap between gene 96 and 97. GeneMark was used to view the coding potential and the results showed that there was no significant coding potential, so the start was not changed. HHPRED produced different results and suggested that gene 96 was a VVR-NUC domain, while NCBI and PhagesDB suggested that gene 96 was a hydrolase.

Future Work:  

The Annotated genes will be reviewed for correctness, and annotations may be changed if needed. After all the genes have been annotated, NapoleonB’s data will be used to test a scientific question.  

Rationale:

The purpose of the lab was to start annotations of NapoleonB’s genes.  

Tools: 

• DNA Master 
• PhagesDB  
• NCBI BLAST 
• Phamerator 
• HHPred 
• GeneMark  
• PhageNotes 

Procedure:  

1. Genes were assigned for phage NapoleonB annotation.  
2. The FASTA file was downloaded and opened with DNA Master.  
3. NapoleonB was autoannotated and the ORF of gene 13 was located.  
4. Gene 13 was annotated on PhageNotes and used information from PhagesDB, NCBI Blast, Phamerator, HHPred, GeneMark,  

Results: 

Gene 13 of NapoleonB had no known function.  

Gene 13 Database Results:

Annotation of Gene 13:

Conclusion: 

Annotation seemed easier with more practice. The start value of gene 13 was not changed and was determined with the support of PhagesDB, HHPred, and NCBI Blast, since gene 13 overlapped with gene 12. The E-value and the coding potential for gene 13 seemed to be better when the start was not changed.  

Future Work :

Annotations of Gene 14,15,16, and 96 will be completed in the future labs.  

Rationale:

The purpose of the lab was to put Elesar’s Gene 44 and 45 annotation into Phage Notes and to learn more about Phamerator and tRNA’s. 

Tools: 

• DNA Master 
• NCBI Blast  
• HHPred  
• Phage Notes 
• PhagesDB – Blast  

Procedure: 

1. An account for Phamerator was created.  
2. The file, which contained annotations of Gene 44 and 45, was opened and annotations were added into Phage Notes.  
3. NCBI Blast, HHPred, and PhagesDB were referenced to confirm that annotations were up to date.  
4. Annotations of Gene 50 and 51 were verified for accurate annotations.  

Results: 

Gene 44 and 45 Annotations : 

• PhagesDB showed that a better match was found for Elesar Gene 44, and therefore changes were made to the annotation.

Conclusion:

More knowledge on annotations was obtained from practice on Gene 44 and 45, and revision of Gene 50 and 51. For annotations to be finished, SIF- synteny and Starterator have to be completed. Skills and knowledge acquired from annotations of Elesar Genes will be used for the annotation of Napoleon B.  

Future Work:  

In the future labs, SIF-Synteny and Starterator will be completed and the process to annotate Napolean B will be started.  

Rationale:

The purpose of the lab was to correct any errors in the genes, and to continue with the remaining annotations of gene 44 and 45.  

Tools: 

• DNA Master 
• NCBI Blast  
• HHPred  
• Gene Mark 

Procedure: 

1. Corrections were made to the annotation of Elesar Gene 44 and 45.  
2. Gene Mark was used to obtain the results for the coding potential of the gene.  
3. DNA Master was opened, and protein sequences were obtained and used in HHPred.  
4. The databases for HHPred were changed according to our phage, and the results were noted.  
5. The genes were blasted on NCBI to obtain the percent alignment. 
6. The annotation was saved to be continued next time.

Results:  

Annotation of Gene 44:  

SSC: 32186, 32380 CP: yes SCS: both BLAST-Start: hypothetical protein PBI_NANDITA_47, 47, NCBI, Q1: S1, 4×10^-23  Gap: 0 LO: N/A RBS: Kibler7, Karlin Medium, 2.942, -2.636, yes SIF-BLAST: NKF, NCBI Blast, Arthrobacter phage Nandita 47, AYN58666.1, 83.33%, 4×10^23. SIF-HHPred : NKF, HHpred, N/A,PF11239.8, 83.33%, 0.65 

Annotation of Gene 45:  

SSC: 32373, 32909 CP: no SCS: both BLAST-Start: hypothetical protein PBI_RYAN_48 , 48, NCBI, Q1: S1, 1×10^-115 Gap: 0 LO: N/A RBS: Kibler7, Karlin Medium, 3.060, -2.325, yes. SIF-BLAST : NFK, NCBI Blast, Arthrobacter phage Ryan 48, AYN59038.1, 54.18%,  1×10^-115 SIF-HHPred : Pertussis toxin S5 subunit, N/A ,PF092.76, 54.18%, 62.95 

Conclusion:

Annotation practice on Elesar Gene 44 and 45 helped to better the understanding of how to annotate. Since HHPred, NCBI, and PhagesDB displayed no known function, it can be concluded that there is no known function for both Gene 44 and Gene 45.

Future Work:

In the future, gene 44 and 45 will be completely annotated and reviewed.  Using this knowledge obtained from Elesar Gene annotations, the process to annotate Napoleon B will be started.

Rationale:

The purpose of the lab was to finish annotating Elesar gene 1 and to practice with two other Elesar genes: 44 and 45.  

Procedure/ Tools: 

1. Elesar gene 1 annotation was opened on DNA Master.  
2. Gaps were determined by the BLAST feature on DNA Master and NCBI.  
3. The frame of gene 1 was opened and the feature RBS provided the RBS scores.  
4. Annotated Gene 44 and gene 45 of Elesar with the same features.  

Results:  

Annotation of Gene 1:

SSC 45,353: CP yes: SCS both-cs: ST: BLAST-Start no significant BLAST alignments: Gap first gene : LO yes: RBS: Kibler7, Karlin Medium, 1.222, -6.751, no  F: SIF-BLAST: SIF-HHPred: SIF-Syn  

Annotation of Gene 44:

• SSC: 32186, 32380 
• CP: yes 
• SCS: both 
• BLAST-Start: [hypothetical protein PBI_NANDITA_47, 47, NCBI, 1, 1, 4×10^-23]  
• Gap: 0 
• LO: N/A 
• RBS: Kibler7, Karlin Medium, 2.942, -2.636, yes 

NCBI results for Elesar Gene 44Annotation of Gene 45:

• SSC: 32373, 32909 
• CP: yes 
• SCS: both 
• BLAST-Start: [hypothetical protein PBI_RYAN_48 , 48, NCBI, 1,1, 1×10^-115] 
• Gap: 0 
• LO: N/A 
• RBS: Kibler7, Karlin Medium, 3.060, -2.325, yes 

NCBI results for Gene 45 

Conclusion: 

More knowledge about DNA Master and Blast was acquired. Practice of annotating gene 44 and 45 improved my skills and understanding of annotating genes. Similar genes were found in the database when Gene 44 and 45 protein sequence was inserted in the database. Very small E values of 1×10-115 and 4×10^-23 were produced indicating that it was not by chance.  

Future Work:  

A deeper and better understanding of the features in DNA Master would be helpful when annotating NapoleonB. More annotations may be done as practice before annotating NapoleonB. 

Rationale: The purpose of the lab was to learn more about BLAST and DNA Master and to start annotating gene 1 of Elesar.  

Procedure/ Tools: 

1. DNA Master was opened and Elesar was auto annotated.  
2. The ORF Frames of Elesar were produced and the start codon of gene 1 was changed.  
3. The protein sequence of Elesar gene 1 used to BLAST on NCBI.  
4. Elesar gene 1 was partially auto annotated until BLAST-Start.  
5. The auto annotations were saved to be continued next time.   

Results: 

The partial auto annotation of Elesar gene 1 was SSC 45,353: CP yes: SCS both-cs: ST: BLAST-Start no significant BLAST alignments.: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn

The Start codon of gene 1 was elongated to 45.  

ORF’s of Elesar and RBS results of Gene 1

Conclusion: 

A better understanding was acquired about Blast and DNA Master. Elesar’s gene 1 protein sequence was inserted into NCBI to be compared to other genes, and the results showed no similar genes to found in the database.   

Future Work:  

In the future, gene 1’s annotation will be finished, and a detailed understanding of DNA Master and auto annotating will be obtained.  

Rationale:

The purpose was to learn about the features of DNA Master specifically about open reading frames using Elesar.  

Tools/Procedure:

1. DNA Master was opened and the code, accessed through file then preference then local settings then new feature, was changed to “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn”.  
2. Elesar was added to DNA Master and Auto Annotated.  
3. The ORF’s of Elesar were observed.  

Results: 

Auto Annotation of Elesar using a Fasta File was obtained using DNA Master. Reverse ORF’s represented by the highlighted portions were viewed.  

Conclusion: 

More features of DNA Master were learnt specifically about ORF’s. Concepts such as reading frames, gaps and overlaps were learnt as well.

Future Work:  

To acquire more knowledge about DNA Master’s features and understand the data presented for Elesar which will aid when annotating NapoleanB.

Rationale/Purpose:

The purpose of the lab was to download and use DNA Master to Auto Annotate Elesar.

Tools/Procedure:

1. DNA Master was downloaded before the class started.
2. Some preferences in DNA Master were changed.
3. Elesar’s FASTA file was obtained off phagesdb.org.
4. The file was added into DNA Master and auto annotated.

Observations/Results/ Conclusions:

DNA Master was downloaded, and the preferences were changed according to the protocols of the lab. Phage Elesar was used to practice and successfully auto annotated using DNA Master. The ORF’s are viewed in the green and the red sections shown below.

In lab I learnt how to navigate DNA Master specifically on how to Auto Annotate using a FASTA file.  

Future Work:

The next step will be to gain a better understanding of some of the features and to be able to interpret the visual results of the Auto Annotations, and to learn how to annotate other phages.