February 12

DNA Day 7

 11 February 2019 ✷ NapoleonB DNA Annotation!

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 the annotation for gene 21 was completed.

Procedure

  • PhagesDB was opened and the FastA file for NapoleonB was downloaded
  • DNA master was opened and NapoleonB was auto-annotated
  • PhageNotes was opened and each aspect of the DNA annotations for genes 21-24 was entered into the program, however, only 21 was annotated to completion and 22 was nearly completed.
  • 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 (this was only done for genes 21 and 22 so far) and both agreed with starterator.
  • The information was recorded and saved in the google sheet.

 

Results

the complete annotation for gene 21 and the partial annotation for gene 22, as entered into PhageNotes, are as follows:

Conclusion

There was some confusion regarding the function of gene 22 of NapoleonB, and time was limited so the annotation wasn’t completed. Further research needs to be completed in order to make an informed decision on the final function of gene 22 because the databases do not agree on the function–PhagesDB and NCBI say it aligns with a tail protein, but HHPred and the conserved domain information both suggest an unknown function related to botox.

Future plans

The function of gene 22 will be further analyzed and researched and the annotation will be finalized. Then, genes 23, 24, and 97 will be annotated.

Category: Lily Goodman | LEAVE A COMMENT
February 12

2/11 ~ Annotation of NapoleonB

Rationale: Annotate genes assigned on Arthrobacter NapoleonB

 

Materials:

  • DNA Mastering
  • PhagesDB Blast
  • NCBI Blast
  • Starterator
  • NCBI Domains Website
  • Computer

Procedure:

  • Opened up DNA Mastering on computer
  • Downloaded NapoleonB file from PhagesDB database
  • Opened the file on DNA Mastering and auto-annotated
  • Was assigned to annotate genes 37-40, placing results on NapoleonB PhageNotes
  • After working on annotations, saved annotated NapoleonB file

Observations:

Genes of NapoleonB!

NCBI Blast results of gene 39

The unfinished annotations of genes 37-40 of Arthrobacter NapoleonB

Conclusion/Next Steps:

  • It’s very exciting to finally start annotating Arthrobacter phage NapoleonB! Although the day was filled with confusion and a little but of chaos, the annotations are on their way to being finished. The next steps would be to continue annotating the genes and then have them be checked.
Category: Justin Yu | LEAVE A COMMENT
February 12

2.11.2019 Annotations of Gene 49 and 50 of NapoleonB

2.11.2019 Annotations of Gene 49 and 50 of NapoleonB

Rationale: Since the procedures and skills for annotating genomes had been developed through extensive practice with phage Elesar, annotations for Napoleon were finally ready to begin. Therefore, genes were assigned and genes 49 and 50 were annotated.

Tools/Procedure:

  • DNA Master
  • Starterator
  • HHPred
  • NCBI BLAST
  • PhagesDB Blast
  • Phamerator
  1. Opened PhagesDB and found NapoleonB fasta file. Downloaded file
  2. Auto-annotated NapoleonB on DNA Master
  3. Opened ORF Screen
  4. Began RBS value examination
  5. Annotated genes 49 and 50

Results:

Gene 49-

 

Gene 50-

 

 

 

Phamerator that applied for both genes:

Gene 49: 

SSC:31823 – 32392, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Tribby gp48 NCBI BLAST q1:s1 0.99 3E-132, Aligns with Arthrobacter Phage Tribby gp48 PhagesDB BLAST q1:s1 0.98 e-100, Gap:15bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.684 -3.22 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF, upstream gene is NKF, downstream gene is NKF, just like in phage Nason

Gene 50: 

SSC:32516 – 32824, CP:No No ORF available would cover all of the potential, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Arcadia gp49 NCBI BLAST q1:s1 0.99 2E-65, Aligns with Arthrobacter Phage Nason gp49 PhagesDB BLAST q1:s1 0.98 e-50, Gap:123bp gap, LO:No, RBS:Kibbler7 and Karlin Medium 3.16 -2.293 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF, upstream gene is NKF, downstream gene is NKF, just like in phage Nason

Conclusions: Both of the genes annotated today had fairly straightforward decisions that were made. The questionable decisions came within the coding potential of gene 50 and the gap that preceded gene 50. There was a very slight amount of coding potential that extended past the earliest start codon of the gene, which resulted in the answer that was listed but there was deliberation on whether or not the amount of potential was considered to be significant. Furthermore, the gap between gene 49 and 50 is slightly longer than is normally seen when examining bacteriophage genomes, so there are some questions about whether or not there is a gene that was not detected by the auto annotation.

Next Steps: The gap between genes 49 and 50 will be examined, and genes 51 and 52 will be annotated during the next available lab time!

Category: Henry Burns | LEAVE A COMMENT
February 11

NapoleonB Annotations 2/11/2019

Rationale: Begin annotating assigned genes of NapoleonB

Process: Used DNA Master, GeneMark, NCBi Blast, NCBi cd, phagesdb, and HHPRED to annotate gene 81 of NapoleonB

  • covered all of coding potential and more so I did not change the start codon
  • had an overlap of 6bp
  • evidence that gene 81 could code for a helicase

Result:

SSC:46764 – 48074, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Circum gp80 NCBI BLAST q1:s1 0.99 0, Aligns with Arthrobacter Phage Circum gp80 PhagesDB BLAST q1:s1 0.99 0, Gap:6bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 1.808 -5.07 No, F:Helicase, SIF-BLAST:ATP-dependent helicase Supported by NCBI BLAST Arthrobacter Phage Circum gp80 ALY08763.1 0.99 0, ATP-dependent helicase Supported by PhagesDB BLAST Circum gp80 KU160642 0.98 0, , SIF-HHPred:Predicted DNA helicase supported by KOG gpNA KOG1016 0.3166 , SIF-Syn:NKF

Next Steps: continue annotating NapoleonB genes 82-84

Category: Rachel Melone | LEAVE A COMMENT
February 11

February 11, 2019 Annotating NapoleonB

Purpose: The purpose of this lab is to begin annotating NaopleonB, specifically gene 93.

Tools/Procedures:

Tools:

  • DNA Master
  • NCBI
  • PhagesDB
  • HHpred

Procedures:

  1. A GeneMark complete sequence was performed on NapoleonB.
  2. Using the information from DNA Master and Starterator, the start and stop were determined for gene 93.
  3. A BLAST search was completed for gene 93 of NapoleonB on PhagesDB and NCBI.
  4. The protein sequence for gene 93 was also run through HHpred.
  5. Based on these results, the annotation for gene 93 was completed and added to Phage Notes for NapoleonB.
  6. The following annotation was recorded: SSC:52622 – 52879, CP:Yes, SCS:Both, ST:SA, BLAST-Start:Aligns with Arthribacterphage Mudcat gp87 NCBI BLAST q1:s1 0.98 2E-53, Aligns with Arthrobacter phage Xenomorph gp88 PhagesDB BLAST q1:s1 1 3E-48, Gap:129bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.802 -4.576 Yes, F:Nicotinamate Mononucleoside Transporter, SIF-BLAST:NKF, SIF-HHPred:Nicotinamide mononucleotide transporter supported by PFam gpNA PF04973.12 0.9302 , SIF-Syn:NKF
  7. The annotations were saved.

Results:
The annotation for gene 93 of NapoleonB was completed. The function for this gene is not certain at this point. The HHpred search matched with the function Nicotinamide mononucleotide transporter, however, this function is not an official function on the SEA-PHAGES list.
Annotation:
SSC:52622 – 52879, CP:Yes, SCS:Both, ST:SA, BLAST-Start:Aligns with Arthribacterphage Mudcat gp87 NCBI BLAST q1:s1 0.98 2E-53, Aligns with Arthrobacter phage Xenomorph gp88 PhagesDB BLAST q1:s1 1 3E-48, Gap:129bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.802 -4.576 Yes, F:Nicotinamate Mononucleoside Transporter, SIF-BLAST:NKF, SIF-HHPred:Nicotinamide mononucleotide transporter supported by PFam gpNA PF04973.12 0.9302 , SIF-Syn:NKF

Conclusion:
Annotations were completed for NapoleonB gene 93 in this lab, but the function of the gene is not yet determined. A possible function was found using HHpred, but as this function is not on the official function list of SEA-PHAGES, it cannot yet be called the function. Further investigation will need to be done to determine the function of this gene. Continuing annotations will lead to a better understanding of each of the resources used and will help in making more accurate calls throughout the annotation process.

Future Work:
Future work will include finishing annotations for NapoleonB, specifically genes 1-4. This will be done in the next lab.

Category: Lucy Potts | LEAVE A COMMENT
February 11

NapoleonB Gene 41 Annotation

Rationale: Begin annotations for NapoleonB genes 41-44. Use knowledge from past few practice annotations to start NapoleonB annotations.

Procedure: NapoleonB FASTa file downloaded from PhageDB, and loaded onto DNA Master. Gene auto-annotated and frames were viewed. NCBI, HHPred, PhageDB, and Phamerator were all pulled up to annotate gene 41. The product of Gene 41 was copied and pasted onto  NCBI, HHPred, PhageDB, and Phamerator. Results were analyzed and annotations were placed into PhageNotes.

Results: 

Gene 41 annotation below followed by NCBI, HHPred, GeneMark, and PhageDB:

SSC: 29246 – 29362, CP: Yes, SCS: Both, ST:SS, BLAST-Start: Aligns with Mudcat gp39 NCBI BLAST q1:s1 2E-18, Aligns with Xenomorph gp38 PhagesDB BLAST q1:s1 1E-14, Gap:2bp gap, LO:N/A, RBS: Kibbler7 and Karlin Medium 3.012 -2.606 Yes, F:NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn:NKF.

Conclusions: Annotation of gene 41 had no known functions.

Future plans: Continue with the annotation of NapoleonB gene 42.

Category: Michael Lum | LEAVE A COMMENT
February 11

02.11.19 Annotating NapoleonB Gene 17

Rationale:

To annotate gene 17 from the phage NapoleonB.

Procedure:

  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 17 was then annotated.

Results:

Gene 17

SSC:12925 – 14769, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Tribby gp18 NCBI BLAST q1:s1 1 0, Aligns with Tribby gp18 PhagesDB BLAST q:s 0, Gap:1bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.486 -4.688 No, F:major tail protein, SIF-BLAST:Tail Protein Supported by NCBI BLAST Tribby gpNA ASR80469.1 1 0, Tail Protein Supported by PhagesDB BLAST Tribby gp18 100 0, , SIF-HHPred:NKF, SIF-Syn:major tail protein, upstream gene is NKF, downstream gene is minor tail protein, just like in phage Nason

Conclusions:

It can be concluded that gene 17 is a major tail protein, as well as identical to the major tail protein found in the phage Tribby.

Next Steps:

The next step will be to annotate genes 18-20 of the phage NapoleonB.

Category: Nathan Newton | LEAVE A COMMENT
February 11

Beginning Annotating NapoleonB Gene 29,30 2/11/19

Rationale: Now that I have learned how to use each Biotech tool I will begin annotating the genome of the phage I discovered the previous semester, along with the help of my classmates. I was tasked with annotating genes 29-32 and 94.

Tools: PhageNotes, DNAMaster, NCBI, PhagesDB, Phamerator, HHPred, Genemark

Procedure:

  1. First loaded the FastA file of NapoleonB into DNAMaster and auto-annotated the genome.
  2. Checked the Frames view in DNAMaster to see if gene 29 was marked at its LORF. Opened the Genemark file for NapoleonB and found that the gene was covering all the coding potential at that area. Noted the start and stop locations and that the coding potential was completely covered.
  3. Using PhagesDB I found that the starterator for gene 29 doesn’t have enough annotated calls to get any information from.
  4. I then BLASTed the product of the gene through the NCBI and PhagesDB databases. Both returned the same result as the best hit. However, the hit didn’t have any result for the function. I then ran the product through HHPred and it didn’t return any hits with a decent probability.
  5. I then calculated the gap of the gene and recorded the RBS results.
  6. I started working on gene 30 and using the Phamerator comparison with other phages in the same cluster, the BLAST results of the gene, and the fact that the gene is a reverse gene in the middle of a forward cassette to determine that the gene needs to be deleted.
  7. Recorded all my findings in PhageNotes.

Results:

BLAST for Gene 29 and Gene 30

Conclusion and Future Work: I found that gene 29 had no known function, and  gene 30 was not really a gene. In the future I will continue working on annotating the genes assigned to me so that we can have a fully annotated genome by the end of the week.

Category: Sriram Avirneni | LEAVE A COMMENT
February 8

Gene 20 and 21 Annotations

2/6/19

Rational:

To enter the annotations made on genes 20 and 21 for Elesar on PhageNotes in order to share it with the rest of the class. This makes it easier and faster to annotate the entire genome of Elesar. Also to check the annotations of two other genes.

Procedure:

  • Entered my name next to the genes I annotated in PhageNotes and added the auto-annotation calls
  • Entered the information for SSC, CP, SCS, LO, BLAST-Start (NBCI), Gap, F, SIF-BLAST (NBCI), SIF-HHPRED, and SIF-Syn
  • BLASTed genes 20 and 21 in phagesdb and entered the needed information
  • For RBS entered only the SD final score, whether it was the best, and the z-score
  • Used the BLAST for phagesdb to predict the function of the genes
  • Checked genes 8 and 9 by annotating them myself and comparing my annotations with the annotation entered

Observations:

  • Information for BLAST-Start and SIF-BLAST using phagesdb was needed
  • For gene 9 the start codon was moved back, but the SCS did not support this change
  • The rest of the annotations for genes 8 and 9 seemed correct

Fig.2 – This image shows the information found on gene 9 from phagesdb. The information matches the annotations entered in PhageNotes.

Conclusion:

The annotations for 20 and 21 were finalized and entered into PhageNotes. Genes 8 and 9 were also checked for mistakes in the annotations. Next lab I will start annotating NapoleonB along with the rest of the class.

Category: Emily Balint | LEAVE A COMMENT
February 8

2-6-19 — Copying Genes 12 and 13 to PhageNotes

Further Annotation of Phage Elesar Genes 12 and 13

Date: 2-4-19

  • Rational
    • The rational for this lab was to annotate the function data for two genes from phage Elesar in order to gain practice in annotation before transitioning to phage NapoleonB.
  • Procedure
    1. DNA Master was opened.
    2. File > Open > Archived DNA Master File was used to open the same Elesar FastA file from before.
    3. Gene 12 was selected through the listing of predicted genes.
    4. The annotation notes were analyzed and copied to the PhageNotes Google Sheets page.
    5. Gene 13 was selected through the listing of predicted genes.
    6. The annotation notes were analyzed and copied to the PhageNotes Google Sheets page.
    7. Using the acquired information, the 12th and 13th genes were annotated.
  • Results
    • The following annotations were copied for Elesar gene 12 :
      • SSC:8224 – 8568, CP:No There is an overlap with the previous gene, SCS:Both, ST:SS, BLAST-Start:Aligns with Nandita gp13 NCBI BLAST q12:s1 0.87 5E-58, Aligns with Phage Ryan gp13 PhagesDB BLAST q12:s2 0.87 1E-48, Gap:7bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.463 -3.67 No, F:head-to-tail stopper, SIF-BLAST:head-to-tail stopper Supported by NCBI BLAST Nandita gp13 AYN58635.1 0.87 5E-58, head-to-tail stopper Supported by PhagesDB BLAST Ryan gp13 MH834627 0.87 1E-48, SIF-HHPred:NKF, SIF-Syn:head-to-tail stopper, upstream gene is head-to-tail adaptor, downstream gene is NKF, just like in phage Ryan
    • The following annotations were copied for Elesar gene 13 :
      • SSC:8565 – 8840, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Nandita gp14 NCBI BLAST q13:s1 0.76 3E-38, Aligns with Phage Nandita gp14 PhagesDB BLAST q13:s2 0.76 7E-31, Gap:4bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 1.137 -7.054 No, F:NKF, SIF-BLAST:NKF Supported by NCBI BLAST gp, SIF-HHPred:NKF, SIF-Syn:NKF
  • Conclusion
    • All the genes present in Phage Elesar have been copied to a single document in order to share the data.
  • Future Plans
    • The next step is to continue annotating more genes on Elesar for practice before transitioning to NapoleonB.
Category: Brandon Reider | LEAVE A COMMENT