February 15

Annotating Gene 14,15,16, and 96 for NapoleonB (2/13/19)

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.  

Category: Sona Subramanian | LEAVE A COMMENT
February 15

Annotation of Gene 13 of NapoleonB (2/11/19)

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.  

Category: Sona Subramanian | LEAVE A COMMENT
February 15

NapoleonB Annotation 2/13/19

Title: NapoleonB Annotation


Date: 13 February 2019


Rationale: The annotations for NapoleonB will be finished and reviewed for accuracy as well as the starterator (ST) annotation completed


Tools:
– Microsoft Surface Pro 5 Tablet
– DNA Master Software
– NCBI BLASTp tool
– HHPred tool
– GeneMark
– PhagesDB BLAST
– Phamerator

– Starterator (PhagesDB)


Procedure: Using the above tools, gene 95 were annotated completely
and the starterator annotation completed for genes 45, 47, and 48.

For gene 95:
– A start codon call was made based on the Glimmer/GeneMark call
– The product sequence was BLAST’ed to determine any present function
– A function no-call was made


Results/Observations: The following picture contains the results of the
annotation for gene 95:

Conclusions/Next Steps: The starterator annotations were finished, giving insight into what information the other phages in the AM cluster revealed about a specific gene’s start. With the exception of one more final review, the genes in NapoleonB have, for the most part, been completely annotated. They will likely be reviewed and any changes made before the analyzation begins on the genome as a whole.

Category: Cooper Johnson | LEAVE A COMMENT
February 15

2/13 ~ Further annotation of NapoleonB

Rationale: Continue annotating the genome of NapoleonB, specifically assigned genes 37-40.

 

Materials:

  • DNA Mastering
  • NapoleonB PhageNotes
  • PhagesDB
  • NCBI Database
  • Phamerator
  • GeneMark

Procedure:

  • Booted up DNA Mastering on computer and opened NapoleonB file
  • Continued annotating NapoleonB genes 37-40
  • After completing, worked on the QTM due that lab day (Number 9)
  • Utilized PhagesDB and NCBI to check protein sequences, and Phamerator to check predicted shape

Observations:

Complete annotated genes of 37-40 for NapoleonB

Questions I had on QTM 9, such as which genes I had problem with

Conclusion/Next Steps: After complete annotation, it’s expected that the genes will be reviewed and checked for correctness. After that process, will continue to work with NapoleonB and create scientific questions to be answered.

Category: Justin Yu | LEAVE A COMMENT
February 15

NapoleonB Annotation 2/11/19

Title: NapoleonB Annotation

Date: 11 February 2019

Rationale: The annotation for NapoleonB has begun and the genome will be sequenced as thoroughly as possible in order to determine function in relation to other similar phages

Tools: 

  • Microsoft Surface Pro 5 Tablet
  • DNA Master Software
  • NCBI BLASTp tool
  • HHPred tool
  • GeneMark
  • PhagesDB BLAST
  • Phamerator

Procedure: Using the above tools, genes 45-48 were annotated completely (with the exception of starterator “ST”). Gene 46 was deleted due to convincing evidence of a bad call. For each gene:

  • A start codon call was made based on the Glimmer/GeneMark calls or lack thereof
  • The product sequence was BLAST’ed to determine any present function
  • A function call was made and any supporting evidence from PhagesDB, Phamerator, and HHPred was logged

Results/Observations: The following picture contains the results of the annotations:

Conclusions/Next Steps: The starterator annotations need to be completed along with gene 95’s complete annotation. All but one gene was labelled as “NKF,” the gene that was able to be identified was an HTH DNA binding domain protein. Once the remaining genes are annotated, NapoleonB will have a completed annotation ready for analyzing.

 

Category: Cooper Johnson | LEAVE A COMMENT
February 14

02.13.19 Annotating NapoleonB Genes 18-20

Rationale:

To annotate genes 18, 19, and 20 of the phage NapoleonB.

Procedures:

  1. DNA Master was opened with the fasta file for NapoleonB being selected.
  2. NapoleonB’s sequence was auto-annotated.
  3. The ORF screen was opened and the RBS values were opened as well.
  4. Gene 18 was then annotated.
  5. Steps 2-4 were repeated for genes 19 and 20 respectively.

Results:

Gene 18:

Gene 19:

Gene 20:

Gene 18:

SSC:14865 – 16796, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with KeaneyLin gp NCBI BLAST q1:s1 0.99 0, Aligns with KeaneyLin gp18 PhagesDB BLAST q1:s1 0.99 0, Gap:95bp gap, LO:No, RBS:Kibbler7 and Karlin Medium 1.803 -5.081 No, F:minor tail protein, SIF-BLAST:Minor Tail Protein Supported by NCBI BLAST KeaneyLin gpNA AXH44156.1 0.99 0, Minor Tail Protein Supported by PhagesDB BLAST KeaneyLin gp18 0.99 0, GDSL-like Lipase/Acylhydrolase supported by a CD found from the pfam database gp pfam13472 NA 1.01E-11, , SIF-HHPred:NKF, SIF-Syn:minor tail protein, upstream gene is major tail protein, downstream gene is NKF, just like in phage Nason

Gene 19:

SSC:16816 – 17196, CP:Yes, SCS:BothGL, ST:SS, BLAST-Start:Aligns with KeaneyLin gp NCBI BLAST q1:s1 0.99 3E-86, Aligns with KeaneyLin gp19 PhagesDB BLAST q1:s1 0.98 6E-69, Gap:19bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.381 -3.86 Yes, F:minor tail protein, SIF-BLAST:Minor Tail Protein Supported by NCBI BLAST KeaneyLin gpNA AXH44157.1 0.99 3E-86, Minor Tail Protein Supported by PhagesDB BLAST KeaneyLin gp19 0.98 6E-69, putative tail-component supported by a CD found from the pfam database gp pfam04883 NA 0.00924, , SIF-HHPred:NKF, SIF-Syn:NKF, upstream gene is minor tail protein, downstream gene is NKF, just like in phage Nason

Gene 20:

SSC:17193 – 21671, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arcadia gp NCBI BLAST q1:s1 0.99 0, Aligns with Nason gp22 PhagesDB BLAST q1:s1 0.99 0, Gap:4bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.535 -4.411 No, F:tape measure protein, SIF-BLAST:Tape Measure Protein Supported by NCBI BLAST Arcadia ASR79986.1 0.99 0, Tape Measure Protein Supported by PhagesDB BLAST Nason gp22 0.99 0, tape measure domain supported by a CD found from the TIGR database gp TIGR02675 NA 0.00000202, , SIF-HHPred:Phage-related tail protein supported by COG gpN/A COG5283 0.2708 1, SIF-Syn:NKF, upstream gene is NKF, downstream gene is NKF, just like in phage Nason

Conclusions:

It can be concluded that gene 18 codes for a minor tail protein as does gene 19. It can also be concluded that gene 20 codes for a tape measure protein. There were no significant challenges in annotating these genes.

Next Steps:

The next step will be to fix any errors found in the annotation of NapoleonB in order for it to be submitted to PhagesDB and subsequently NCBI.

Category: Nathan Newton | LEAVE A COMMENT
February 14

DNA Day 8

 13 February 2019 ✷ NapoleonB DNA Annotation pt 2

Rationale:  NapoleonB’s genes were assigned to each student to annotate and record in order to complete the genome annotation for the phage. Annotations for genes 21-24 and 97 were started and completed.

Procedure

  • DNA master was opened and NapoleonB was auto-annotated
  • PhageNotes was opened and each aspect of the DNA annotations for genes 22-24 was entered into the program and genes 22-24 and 97 were annotated to completion.
  • Blasts for products (protein) were run on PhagesDB, HHPred, and NCBI to ensure accurate and up-to-date blast information.
  • Phamerator was opened for AM phages and the synteny of NapoleonB was analyzed compared to genomes of phages in the AM group.
  • From PhagesDB, starterator for each gene was opened and compared and all genes annotated agreed with starterator.
  • The information was recorded and saved in the google sheet.

 

Results

the complete annotations for genes 22-24 and 97 as entered into PhageNotes, are as follows:

 

 

Conclusion

In genes 22 and 23, there was uncertainty in the function of the gene. The databases couldn’t agree or provide a function, thus it was decided that the function was unknown, or NKF.

Future plans

The annotation of NapoleonB’s genome will be completed, checked, and submitted to phagesdb. The information gathered from the annotation of the genome will lead to the creation of a research question and thus further research into phage biology involving NapoleonB.

Category: Lily Goodman | LEAVE A COMMENT
February 14

NapoleonB Genes 26-28 (2/13/19)

Rationale:

Annotate NapoleonB genes 26-28 to complete the section of genes assigned. Once NapoleonB genome is fully annotated, ideas for what research question should be investigated with this data can be explored.

Tools:

  • DNA Master
  • NCBI
  • PhagesDB
  • GeneMark
  • HHpred
  • Phamerator
  • phage notes

Procedure:

  1. Conducted a GeneMark on NapoleonB.
  2. Determined the stop and start with the highest coding potential for genes 26-28 based off the “Choose ORF Start” window and the GeneMark.
  3. Performed a HHpred search on NapoleonB genes 26-28.
  4. BLAST was performed on NapoleonB genes 26-28 through both NCBI and PhagesDB.
  5. Annotated NapoleonB genes 26-28.

Results:

The following image shows NapoleonB gene 26-28’s annotations.

The following images show the BLAST E-values for gene 26 from the start codon which Glimmer and GenemMark called.

The following images show the BLAST E-values for gene 26 from the new start codon which has the longest reading frame.

Conclusion:

On gene 26, it was determined that the start codon should be moved from what Glimmer and GeneMark called (bp 24817) to an earlier start codon (bp 24802) because the BLAST E-values were smaller, more coding potential would be covered, and this would make the gene have the longest open reading frame. Genes 26-28 had no known function.

Future Work:

NapoleonB’s annotations will be checked and research questions to be investigated will be brainstormed.

Category: Kathryn Adkins | LEAVE A COMMENT
February 14

Annotation of NapoleonB Part Two 2/13/2019

Annotation of NapoleonB Part Two 2/13/2019

Rationale

The rationale behind these procedures is to start annotations on NapoleonB so that other research can be conducted.

Tools/Procedure

  1. Previous DNA Master Annotations were loaded
  2. Reading frames were examined (Genome > Frames> click “ORFS” button)
  3. Gene locations were compared paying attention to gaps and overlap
  4. NCBI was used to BLAST amino acid sequences against a large database of recorded sequences
  5. Phages DB was used to BLAST amino acid sequences against the SEAPHAGES database
  6. HHPred was used to compare the hypothetical protein structure against other hypothetical structures
  7. Starterator and genemark were used to determine if the genes ought to be altered to include more or fewer base pairs
  8. The information gathered was used to update phage notes

Results

Gene 65 – SSC:41757 – 41909, CP:Yes, SCS:Glimmer, ST:SA, BLAST-Start:Aligns with Arthrobacter phage Cheesy gp63 NCBI BLAST q1:s1 0.87 7E-22, Aligns with Arthrobacter Phage Cheesy gp63 PhagesDB BLAST q1:s1 1 7E-20, Gap:24bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 3.269 -2.511 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Gene 66- SSC:41909 – 42091, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter phage Cheesy gp64 NCBI BLAST q1:s1 0.86 2E-24, Aligns with Arthrobacter Phage Kardesai gp65 PhagesDB BLAST q1:s1 1 4E-27, Gap:1bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.433 -4.402 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Gene 67- SSC:42238 – 42366, CP:No No avaliable start codon, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter phage Mudcat gp62 NCBI BLAST q1:s1 1 3E-20, Aligns with Arthrobacter Phage Mudcat gp62 PhagesDB BLAST q1:s1 1 7E-17, Gap:146bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 3.269 -2.064 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Gene 68- SSC:42368 – 42739, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Circum gp68 NCBI BLAST q1:s1 0.95 3E-76, Aligns with Arthrobacter Phage Kardesai gp67 PhagesDB BLAST q1:s1 0.97 8E-61, Gap:1bp gap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.433 -3.829 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Conclusion

There is very little information from which to draw conclusions about the data set; however, it appears that there is no known function for genes 65 through 68. As the rest of Napoleon B is annotated I will be able to draw more conclusions from the data.

Future Plans

In the future, I will continue my annotations of NapoleonB with the goal of being able to research my own questions once the gene is fully annotated.

Category: Uncategorized | LEAVE A COMMENT
February 14

NapoleonB Gene 25 (2/11/19)

Rationale:

Start annotating NapoleonB. Once NapoleonB fully genome is annotated, the process of brainstorming research questions about NapoleonB can begin.

Tools:

  • DNA Master
  • NCBI
  • PhagesDB
  • GeneMark
  • HHPred
  • Phamerator
  • phage notes

Procedure:

  1. Auto-annotated NapoleonB FASTA file.
  2. Conducted a GeneMark on NapoleonB.
  3. Determined the stop and start with the highest coding potential for gene 25 based off the “Choose ORF Start” window and the GeneMark.
  4. Performed a HHPred search on NapoleonB gene 25.
  5. BLAST was performed on NapoleonB gene 25 through both NCBI and PhagesDB.
  6. Annotated NapoleonB gene 25.

Results:

The following image shows NapoleonB gene 25’s annotation.

The following images were examined to determine whether or not there was enough supporting evidence for a function.

HHPred Results

NCBI BLAST Results

CDD Results

BLAST PhagesDB Results

Phamerator Comparison

Conclusion:

A difficult call occurred when determining whether or not there was enough supporting evidence for gene 25 to have a function. HHPred called a holin function with a 99.92 probability. CDD also called the holin function. Both BLAST results showed a low percent alignment, but the E-values were acceptable. On Phamerator, none of the other AM cluster genes have the holin function labeled on them. Gene 25 was annotated with the holin function.

Future Work:

Annotate NapoleonB genes 26-28.

Category: Kathryn Adkins | LEAVE A COMMENT