February 15

Annotation of Genes 34-36 in NapoleonB (2/13/19)

Rationale:

Upon the completion of the annotation of gene 33, genes 34-36 were annotated with the usage of a variety of programs and databases.

 

Tools:

  • Personal Computational Device
  • DNA Master
  • NCBI
  • PhagesDB
  • GeneMark

 

Results:

  • After the annotation of gene 33 was checked, the annotation of gene 34 began after the gene was viewed on DNA Master to determine if it had any gaps or overlaps.
  • Because there was a gap, a better reading frame to minimize the gap was attempted to be found but the original was the largest open reading frame.
  • From the databases, evidence was gathered to prove the function which showed to have no known function.
  • The annotation of gene 34 was shown to be

  • After the completion of 34, gene 35 was viewed on DNA Master and it was seen that the best open reading frame was not chosen
  • To change the frame, the 5′ base pair was changed to obtain the best frame.
  • After the change, the coding potential was viewed which showed that there was no coding potential regardless if the best or original reading frame was chosen.
  • The annotation of gene 35 was shown to be

 

  • Gene 36 was then annotated with the same process that was used to determine the annotation of gene 34.

 

 

Conclusion:

For this lab, genes 34-36 were annotated and gene 33 was checked. The 5′ end of gene 35 had to be changed to fit the best open reading frame and then the coding potential was viewed on GeneMark. Other than this difference, all the genes required evidence to prove that the function was what it was which was no known function for all the genes.

Future:

Because the annotation for all the genes have been completed and checked, individual research questions will be thought of for the individual projects.

Category: Sabin Patel | LEAVE A COMMENT
February 15

Annotating NapoleonB Gene 33 (2/11/19)

Rationale:

Genes from NapoleonB were assigned and the rest of the lab was given to annotate the four assigned genes.

 

Tools:

  • Personal Computational Device
  • DNA Master
  • NCBI
  • PhagesDB
  • GeneMark

 

Results:

  • After the genes were assigned, gene 33 was viewed on DNA Master to determine if the opening reading frame had the most coding potential if the second biggest opening reading frame had the most coding potential.
  • To determine this, the base pairs for the second opening reading frame was changed and viewed on GeneMark and compared with the coding potential of the original open reading frame. It was concluded that the coding potential for the original open reading frame was best because it covered all the coding potential

 

  • The opening reading frame was reverted back to its original frame and the product was used to compare the sequence with other sequences in databases such as NCBI, HHPred, and PhagesDB.
  • With these databases, other parts of the annotations were determined in order to form the complete annotation.
  • The annotation for gene 33 was

 

Conclusion:

Upon the assignment of the genes of 33-36, the annotation of gene 33 began. Because there was an overlap between gene 32 and 33, the open reading frame was shifted to the second best; however, it was determined that the original reading frame was the best because it covered all the coding potential, unlike the second reading frame. After the best reading frame was determined, the different parts of the annotation were found from databases such as NCBI, GeneMark, HHPred, PhagesDB, and DNA Master. The function of gene 33 was found to have no known function by NCBI, HHPred, and PhagesDB. The annotation for gene 33 was completed.

Future:

In the future (2/13/19), genes 34-36 are to be annotated and annotation of gene 33 is to be checked.

 

Category: Sabin Patel | LEAVE A COMMENT
February 15

Annotation of Genes 9 and 10 for NapoleonB

2/11/19

Rational:

To start annotating the assigned genes 9-12 and 98 for NapoleonB and enter them in PhageNotes

Procedure:

  • Dowloaded the fastA and auto-annotated NapoleonB
  • Entered the gene call after genes 9 and 10 in the home tab for PhageNotes
  • Downloaded the genemark for NapleonB
  • Checked to see if the coding potential was covered by the current start and that it was the ongest ORF
  • Entered the start and stop codon
  • Entered who the start was call and chosen by
  • Checked phagesdb to see whether the gene’s start matches with the most called start (starterator)
  • BLASTed genes 9 and 10 through NBCI and Phagesdb
  • Ran the genes through HHpred
  • Entered the required information for the NBCI and phagesdb hit in PhageNotes
  • Calculated the gap/overlap for the gene and its upstream gene
  • Selected the gene to get the information needed for RBS
  • Entered the information for the hits in NBCI (BLAST and CDD), Phagesdb, and HHpred for the function
  • BLASTed the surrounding genes to look for for SIF-Syn

Observations:

  • The genemark showed that the current start covers all the coding potential for genes 9 and 10
  • Both genes were LORF
  • Gene 9 had a gap of more than 10 bp
  • Genes 9 and 10 both agreed with the starterator
  • For gene 9 the NBCI hit was Arcadia and phagesdb was Tribby
  • For gene 10 the NBCI hit was Mudcat and phagesdb was Xenomorph
  • Gene 9 had a gap of 14 and gene 10 had an overlap of 4
  • For both genes the SD score for the chosen start was the best
  • The funtion for gene 9 was portal protein and gene 10 was hydrolase

Conclusion:

The genes 9 and 10 were finished and there function were found to likely be a portal protein and a hydrolase repectively. Note the only source that supported gene 10 as a hydrolase was a more specific type of hydrolase. Next lab I will finish the annotation of 11, 12, and 98.

Category: Emily Balint | LEAVE A COMMENT
February 15

2/13 NapoleonB Annotations

Purpose and Rationale: Finish annotating the genes assigned to me, proofread them, and assist other people with their genes.

Procedure:

  • Proofread genes 73-76.
  • Marked all genes as ok to check
  • Helped Rachel find the starting point to gene 83

  • Nothing else to help with, so ended lab time for the day

Results: All 4 of my genes are completed, as well as the majority of the rest of NapoleonB’s genes.

Future Plans: Review the genome. Search through the genome for any interesting patterns, remarkable findings, etc.

Category: Uncategorized | LEAVE A COMMENT
February 15

Continuing Annotating Genes 86-88 of NapoleonB 2/13/19

Rationale

Today we will finish the annotations assigned for the genome of NapoleonB.

Tools

  • NCBI
  • HHPred
  • Phamerator
  • PhagesDB
  • Genemark
  • DNA Master
  • Phage Notes

Procedure

  • DNA master was opened and NapoleonB’s FASTA file was autoannotated.
  • The protein sequences were run through NCBI Blast, HHPred, and PhageDB.
  • The remaining tools were utilized to complete the rest of the annotations of Phage Notes. Phamerator was utilized to determine the synteny, while genemark displayed the coding potential.

Results

The results for genes 86-88 are shown below.

Conclusions/Next Steps

Next, we will ensure that no errors were detected in the annotated genome. The annotations will then be submitted to PhagesDB.

Category: Emily Gaw | LEAVE A COMMENT
February 15

Annotating Genes 85-88 of NapoleonB 2/11/19

Rationale

Today we started annotating the genome of NapoleonB.

Tools

  • DNA Master
  • NCBI Blast
  • HHPred
  • PhagesDB
  • Genemark
  • Phamerator
  • PhageNotes

Procedure

  • NapoleonB’s FASTA file was downloaded and opened in DNA Master.
  • The genome as auto-annotated.
  • Gene 85 was located and DNA Master, NCBI Blast, HHPred, and PhagesDB were utilized to help annotate the genome on PhageNotes. Genemark and Phamerator were utilized as well.

 

Results

The results of the final annotation of gene 85 is shown below. The function of gene 85 was reported to be HNH endonuclease.

Conclusion/Next Steps

Next, the annotations of genes 86-88 will be conducted to finish the assigned genes of Napoleon B. The annotations will allow for further research on NapoleonB,

Category: Emily Gaw | LEAVE A COMMENT
February 15

02/13/19 Finishing NapoleonB Genes 63-64

Rationale: 

The purpose of today’s lab was to finish annotating the final 2 genes assigned in NapoleonB

Tools:

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

Procedure:

  • Lab began immediately with finishing up final annotations of assigned genes from the previous lab.
  • Ran genes 63 and 64 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 63:
    • SSC:41221 – 41427, CP:Yes, SCS:BothGM, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Arcadia gp62 NCBI BLAST q1:s1 0.95 2E-32, Aligns with Nason gp62 PhagesDB BLAST q1:s1 0.96 1E-28, Gap:4bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 1.104 -6.698 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF supported by gp , SIF-Syn:NKF
  • NCBI BLASTp Results:
  • PhagesDB BLASTp Results:
  • HHPred Results:
  • Phamerator Results:
  • Final Annotation for Gene 64:
    • SSC:41417 – 41782, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arhtrobacter Phage Correa gp60 NCBI BLAST q1:s1 0.64 1E-46, Aligns with Arthrobacter Phage Tribby gp65 PhagesDB BLAST q1:s1 0.76 1E-38, Gap:9bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 3.16 -2.354 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF supported by gp , SIF-Syn:NKF
  • NCBI BLASTp Results:
  • PhagesDB Results:
  • HHPred Results:
  • Phamerator Results:

Conclusions:

For both genes, the protein call was decided to be NKF as there was not enough supporting information to call a protein function with confidence. Despite there being a potential hit on HHPred, the probability was not enough to be sufficient for a call. Also the RBS for gene 63 despite having a low z-score was chosen as other suggested RBS shortened too much of the gene.

Next Steps:

The next steps for this lab are to edit and finalize any errors in the gene annotations made.

 

 

Category: Gabriel Andino | LEAVE A COMMENT
February 15

NapoleonB Annotation

Purpose and Rationale: Annotate Phage NapoleonB

Procedure:

  • Downloaded the NapoleonB fasta file
  • Auto-annotated NapoleonB in DNA Master

 

  • Began with Gene 73
  • Confirmed called start and spot with Glimmer and Genemark, and RBS computations
  • Compared coding potential to Genemark Heuristics map. Gene 73 has about 30 base pairs left without a start point, making it inaccessible
  • the next step was to BLAST the amino acid structure in NCBI and PhagesDB
  • Phagesdb had several hits, but none that had a defined function
  • NCBI showed similar results, showing, for the most part, the same phages as in PhagesDB
  • HHPred was able to place the protein in a family, but again the family had no function
  • Without a function for the protein, there is no way to predict synteny
  • Phamerator shows similar genes in similar places for phages in the same family as NapoleonB, all without known functions as well

Repeated all steps above with Genes 74, 75, and 76.

Upon Completion:

  • Reviewed each gene and ensured everything was filled out properly
  • Marked all four genes as complete in Phagenotes

Results: All four of my genes are annotated to the best of my ability. There isn’t a whole lot to do, because there are no similar genes that code for proteins with known functions. There are some interesting notes to take out of the information presented.

Observations:

  • All four genes have no known function.
  • The gaps separating the genes are very short, but they are all gaps. The longest gap is 24 base pairs.

From HHPred:

  • The proteins coded by genes 73 and  74 were placed in the same family, but the family has no known function.
  • The protein from gene 75 was also placed in a different family, and again in one without a known function
  • Gene 76 codes for a protein, but there were not enough hits to place the protein in a family

Function:

  • NCBI and PhagesDB were unable to locate any similar proteins with known functions for the proteins coded by genes 73-76
  • No other method of prediction was able to determine any evidence of function
  • Despite lack of function, these genes were highly similar to genes found in many other Arthrobacter phages, from across the eastern and central United States

Future Plans: I understand that there are a lot of people who are having more trouble completing their annotations than I am. I can either help take on excess genes, or begin proofreading through their annotations to ensure their accuracy. It all depends on what is needed at the beginning of next lab session.

Category: Uncategorized | LEAVE A COMMENT
February 15

02/11/19 NapoleonB Annotations

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.

 

Category: Gabriel Andino | LEAVE A COMMENT
February 15

NapoleonB Annotations 2/13/19

Rationale: Continue annotating assigned genes of NapoleonB

Process: Used DNA Master, GeneMark, NCBi Blast, NCBi cd, phagesdb, and HHPRED to annotate gene 82-84 of NapoleonB

  • changed the start codon of gene 83 to include more coding potential
  • no evidence of any function for all three genes

Result:

Gene 82:

SSC:48102 – 48746, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter phage KeaneyLin gp77 NCBI BLAST q1:s1 0.99 2E-145, Aligns with Arthobacter Phage KeaneyLin gp77 PhagesDB BLAST q1:s1 0.96 1E-114, Gap:27bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 3.263 -2.348 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Gene 83:

SSC:48743 – 48818, CP:Yes, SCS:BothGM, ST:SS, BLAST-Start:Aligns with Arhtrobacter Phage Circum gp82 NCBI BLAST q1:s1 0.76 1E-32, Aligns with Arhtobacter Phage Circum gp82 PhagesDB BLAST q1:s1 76%% 2E-28, Gap:4bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.188 -4.267 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Gene 84:

SSC:49015 – 49233, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter phage Cheesy gp84 NCBI BLAST q1:s1 0.98 4E-45, Aligns with Arthrobacter Phage Cheesy gp84 PhagesDB BLAST q1:s1 0.98 3E-36, Gap:65bp gap, LO:, RBS:Kibbler7 and Karlin Medium 1.98 -4.847 No, F:NKF, SIF-BLAST:NKF Supported by NCBI BLAST gp , NKF Supported by PhagesDB BLAST gp , , SIF-HHPred:NKF, SIF-Syn:NKF, upstream gene is , downstream gene is , just like in phage

Next Steps: continue annotating NapoleonB genes

Category: Rachel Melone | LEAVE A COMMENT