Rationale: 

The purpose of today’s lab was to take all of the tools given to us in our bioinformatics toolkit and finally apply them to the annotation of NapoleonB.

Tools:

• DNA Master
• NCBI BLASTp
• HHPred
• Phamerator
• Starterator
• PhagesDB BLASTp
• Genemark (pdf)

Procedure:

• Lab began with a review of all the tools in the bioinformatics toolkit.
• Genes for NapoleonB were split up and assigned to be annotated.
• Genes 61-64 were assigned to me to be fully annotated.
• Ran genes 61 and 62 through a series of BLASTp searches in both NCBI and PhagesDB, searched for protein folding structure in HHpred, analyzed the starterator analysis through PhagesDB, and checked for any supporting information of synteny by examining similar phages in the AM cluster with NapoleonB.

Results:

• Final Annotation for Gene 61:
  • SSC:36690 – 37253, CP:Yes, SCS:BothGL, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Mudcat gp57 NCBI BLAST q2:s1 0.99 3E-17, Aligns with Mudcat gp57 PhagesDB BLAST q2:s1 0.99 1E-108, Gap:14bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 3.118 -2.454 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF
• BLASTp Results:
  • 
  • PhagesDB BLASTp Result matched with the same phage and gene as the BLASTp result
• HHpred Result:
  • 
• Phamerator Result (Top gene is NapoleonB in comparison to bottom gene from Nason):
  • 
• Final Annotation for Gene 62:
  • SSC:37322 – 41224, CP:Yes, SCS:BothGM, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage KeaneyLin gp60 NCBI BLAST q1:s1 0.99 0, Aligns with KeaneyLin gp60 PhagesDB BLAST q1:s1 0.99 0, Gap:68bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.905 -3.803 No, F:DNA primase/polymerase, SIF-BLAST:DNA Polymerase Supported by NCBI BLAST KeaneyLin gp60 AXH44198.1 0.99 0, DNA Polymerase Supported by PhagesDB BLAST KeaneyLin gp60 0.99 0, Phage or plasmid associated DNA primase supported by a CD found from the COG databse prophage gp COG3378 2.9E-13, , SIF-HHPred:DNA Polymerase Delta supported by KOG gp KOG0969 0.8194 , SIF-Syn:NKF

• BlastP Result:
  • 
  • PhagesDB BlastP result was identical
• HHpred Result:
  • 
• Phamerator Synteny (Mudcat Top, NapoleonB bottom):
  • 

Conclusions:

• For Gene 61:
  • Despite yielding a BLASTp result with high probability, there was not enough supporting information to help declare a function. Due to this, a function of NKF was called for this hypothetical protein. Calling the start was difficult, but decided to agree with the glimmer start to pull the gene back as far as possible to give the longest open reading frame.
• For Gene 62:
  • There was a significant amount of information to support the calling of a function for the protein examined. NCBI BLASTp and HHPred both gave the function as a DNA Primase/Polymerase, and the synteny from examining Arthrobacter Phage Mudcat seems to support the call. HHPred called a DNA Polymerase Delta found in Eukaryotic cells, which also supports the assumption that the structure of the protein coded in gene 62 is almost identical to DNA Polymerase Deltas found in eukaryotic cells.

Next Steps:

The next steps for this experiment are to continue with the annotation of NapoleonB genes and finish the final two genes in the following lab.