January 31

Elesar Gene 54 & 56 (1/30/19)

Rationale: Finish going over genome annotation temple and practice annotating with Elesar gene 54 and 56.

Tools:

  • DNA Master
  • NCBI
  • PhagesDB
  • GeneMark

Procedure:

  1. The BLAST-Start, Gap, LO, and RBS portions of Elesar gene 1 were filled out.
  2. BLAST was performed on Elesar gene 54 through both NCBI and PhagesDB.
  3. Annotated Elesar gene 54.
  4. BLAST was performed on Elesar gene 56 through both NCBI and PhagesDB.
  5. Annotated Elesar gene 56.
  6. These annotations were saved.

Results:

The following image shows Elesar gene 1 fully annotated.

The following images show the GeneMark for Elesar between 36000 to 40000 bp.

The following images show the BLAST results for Elesar gene 54.

The following image shows Elesar gene 54 fully annotated.

The following images show the BLAST results for Elesar gene 56.

The following image shows Elesar gene 56 fully annotated.

Conclusion:

For both Elesar genes 54 and 56, they were most similar to another gene from Elesar. PhagesDB seems to have more BLAST results with smaller E values. Although PhagesDB is a smaller database, it seems to be more helpful than the NCBI database since it has more phage DNA.

Future Work:

Continue to practice annotating with Elesar and learn about phamerator.

Category: Kathryn Adkins | LEAVE A COMMENT
January 30

BLAST (1/28/19)

Rationale: Go over guiding principles along with other analysis tools, such as GeneMark and BLAST and start to fill in the notes template.

Tools:

  • DNA Master
  • NCBI
  • PhagesDB
  • GeneMark

Procedure:

  1. In DNA Master, the main window, the frames window, and the choose ORF start window were opened.
  2. BLAST was performed on Elesar gene 1 using both NCBI and PhagesDB.
  3. The SSC, CP, and SCS portions of the template for Elesar gene 1 were filled out.
  4. This annotation was saved.

Results:

After performing BLAST through both NCBI and PhagesDB, it was determined from the high E value that there was no significant alignment found. The images below show these results.

It was determined through GeneMark that the start choice (84 bp) predicted by Glimmer and GeneMark was not the LORF (longest open reading frame). At 45 bp, all possible coding potential was covered as shown in the GeneMark graph below.

The following screenshot shows what parts of the template where filled in along with the values in the choose ORF start window.

Conclusion:

Although it is rare to see TTG as a start codon, it can be a start codon. The start choice predicts from Glimmer and GeneMark are not always accurate.

Future Work:

The result of Elesar gene 1 template will be filled out.

Category: Kathryn Adkins | LEAVE A COMMENT
January 30

Elesar Annotations

Purpose and Rationale: Annotate Phage Elesar to find the proper starts, stops, and other features to the genes. Annotating these genes will help us refine the process of annotation and improve mastery of the computer program.

Procedure:

  • Opened Elesar dnam5 file.
  • Continued adding annotation notes to gene 1.
  • Assigned genes 26, 27 to complete annotations on.
  • Found start/stop of gene 26
  • Determined that the start and stops matched the coding potential found in Genemark.

Elesar Gene 26, showing the beginning of coding potential at the same base as the start codon, 18815, and stopping pair 19690.

  • Blasted gene in both databases. Found to be most similar to a bacterial peptidase, known as M23.

This particular peptidase is found in Arthrobacter sp. KI72, potentially raising a “chicken or the egg” question

  • Continued to fill in the description notes with the remaining information

Began work on gene 27

  • Found the auto-annotated start and stop, and that they were accurate with the coding potential and the stop codon.
  • Coding potential continued after the end of gene 27, into gene 28, however, the stop codon provides the end of the gene.

Note the gap just after base pair 20,000, where the coding potential dips but does not hit zero. It flows right into gene 28.

  • Blasted the Gene, and found it to be similar to bacterial proteins without a known function.

  • Finished writing genetic notes into the description

Results: Completed annotating all portions of Genes 26 and 27 that had been taught to us. There are other portions of the notes left to be completed, but that will have to wait until we are instructed on what to do.

Future Plans: Continue Annotations as instructed next lab period.

 

Category: Uncategorized | LEAVE A COMMENT
January 30

1.30.19 Annotating Elesar Genes

1.30.19 Annotating Elesar Genes

Rationale: Today, two genes were annotated from the Elesar genome to begin practicing the skill of completely annotating a gene from a bacteriophage genome.

Procedure:

  • Opened FASTA file with previous example annotations on Gene #1
  • Answered Gap, LO, and RBS.
  • Assigned genes 50&51.
  • Annotated genes 50&51 from the Elesar genome.

Results:

 

Gene 50

SSC (Start/Stop Coordinates): 36095,36970

CP (Coding Potential): Yes

SCS (Start-choice source): Both.

BLAST-Start: Nandita, gp50, NCBI, Query 1 to Subject 1, 99%, 0.0.

Gap: 40.

LO (Longest ORF): Yes.

RBS (Ribosome Binding Site): Kibler7,Kibler Medium, 2.097, -4.366, Yes.

Gene 51

SSC: 36967,37350
CP: Yes
SCS: Both.
BLAST-Start: Auxilium, gp 51, NCBI, Query 1 to Subject 1, 96%, 4e-14.

Gap: 4bp overlap.

LO: N/A

RBS: Kibler7, Kibler Medium, 2.496, -3.662, Yes.

Conclusions: After performing the annotations for both gene 50 and 51, new skills were developed and a slightly better understanding of gene annotation was achieved. A complete understanding of the process has yet to be obtained, but as more genes are practiced, it will hopefully become more clear in regards to what to do for each step.

Future Steps: In the future, more genes will be practiced on Elesar to develop additional skills, but NapoleonB will be annotated eventually when confidence in the skills has been developed.

Category: Henry Burns | LEAVE A COMMENT
January 30

1.30.19 Elesar Annotation

Rationale:

To continue practicing genome annotation with DNA Master by annotating two genes of Elesar’s genome.

Procedures:

  1. Used an already opened and auto-annotated copy of Elesars genome on DNA Master.
  2. Annotated genes 48 and 49 of Elesar.

Results:

Gene 48: SSC: [34620, 35063], CP: yes, SCS: Both, BLAST-Start: Elesar, gp45, PhagesDB, Query 1 to Subject 1, 100%, 6e-83 Gap: 113bp gap, LO: yes, RBS: Kibler7, Karlin Medium, 2.611, -3.929, no

Gene 49: SSC: [35215, 36054], CP:yes, SCS: Both, BLAST-Start: Nandita, NCBI, Query 1 to Subject 1, 95%, 8e-165, Gap: 151bp gap, LO: yes, RBS: Kibler7, Karlin Medium, 2.970, -3.344, yes

Conclusions:

From the results gathered it can be concluded that I learned more about how to annotate genomes on DNA Master.

Next Steps:

The next step will be to continue practicing with DNA Master in annotating Elesar’s genome in order to eventually annotate the genome of the phage NapoleonB.

Category: Nathan Newton | LEAVE A COMMENT
January 30

Lab Day 4: Gene 4 and 5 Annotations + BLAST

Rationale

Annotate and update template for genes 4 and 5 from elesar. Practice learning how to annotate each gene.

Procedure

  1. Opened DNA master with elesar fasta file
  2. Opened DNA frames, clicked on ORFs, highlighted target gene, clicked on RBS
  3. Blasted gene 4 via phagedb
  4. Filled out template for SSC, CP, SCS, Blast-start, Gap, LO, and RBS and used guidelines and annotation key for assistance
  5. Repeated same procedures above for gene 5.
  6. Clicked post after each gene annotation and saved as new dnam5 file.

Observations

Both gene 4’s and 5’s scores are not the lowest, which is why RBS has “no” on both of them, but there is still a high chance that both genes coded for Nandita.

Next Steps

Finish the rest of the template and have a better understanding of annotating any gene from elesar to prepare the annotation of NapoleonB.

Category: Soo-Un Jeong | LEAVE A COMMENT
January 30

January 30 2019 Annotating Genes 52 and 53

Purpose: The purpose of this lab was to learn more about annotations and to practice annotation on genes of Elesar.

Tools/Procedures:

Tools:

  • DNA Master
  • GeneMark
  • NCBI
  • PhagesDB
  • Annotation Guide

Procedures:

  1. A GeneMark complete sequence was performed on Elesar
  2. A BLAST was performed on Elesar gene 52 with NCBI and PhagesDB and the results were compared.
  3. Using the annotation guide, the annotations for gene 52 were complete, excluding ST and ending after RBS. The information needed for the annotation came from DNA Master, NCBI, PhagesDB, and GeneMark.
  4. The following annotation was recorded on DNA Master for Elesar Gene 52. SSC:37347,37562 CP:no, no start SCS:both ST: Blast-start:Ryan,57,NCBI,1:1,98%,6e-30 Gap:4 LO:NA RBS:Kibler7,Karlin Medium,2.657,-3.320,no
  5. A  BLAST was performed on Elesar gene 53 with NCBI and PhagesDB and the results were compared.
  6. Using the annotation guide, the annotations for gene 53 were complete, excluding ST and ending after RBS.
  7. The following annotation was recorded on DNA Master for Elesar Gene 53. SSC:37559,37732 CP:no, no start SCS:both ST: Blast-start:Elesar,50,PhagesDB,1:1, 100%, 7e-23 Gap:4 LO:NA RBS:Kibler7, Karlin Medium,2.893,-2.740,no
  8. The annotations were saved.

Results:

Annotations for Elesar genes 52 and 53 were begun. The start and stops were chosen, and the genes were compared to other genes on NCBI and PhagesDB.

Conclusions:
Continuing the process of learning how to manually annotate genes will aid in the process of annotating NapoleonB’s genes. The two genes annotated were not complete, but the process was begun. Both of the genes annotated matched with another protein that had very low e values. Both of the genes were also found to have overlaps with other genes. Interestingly, the BLAST results showed that Elesar Gene 53 matched with Elesar gene 50 on PhagesDB. This could be due to manual annotations that were done previously on Elesar.

Future Work:
Future work will include learning the rest of the annotations and continuing to practice annotations. This practice will eventually help in the process of annotating NapoleonB.

Category: Lucy Potts | LEAVE A COMMENT
January 30

1/30 ~ Elesar Practice Annotations

Rationale: Practice annotations of Elesar. Assigned two genes to completely annotate

Materials:

  • Computer
  • NCBI Database
  • PhagesDB Database
  • GeneMark
  • DNA Mastering

Procedure:

  • Opened saved DNA Mastering from previous lab
  • Annotated genes 24 and 61 for Elesar
  • Focused on SSC, CP, SCS, BLAST-Start, GAP, LO, and RBS
  • Used GeneMark to determine if the gene had coding potential

Observations:

Coding potential of gene 61 of Elesar

Coding potential of Gene 24 of Elesar

Annotations of Gene 61

Annotations of Gene 24

The annotations of Genes 24 and 61, completed for QTM 5

Conclusion/Next Steps: This practice allowed as an example for when NapoleonB is annotated.

Category: Justin Yu | LEAVE A COMMENT
January 30

Annotating Elesar Gene 28,29 1/30/19

Rationale: Continuing to practice annotating I will be annotating genes 28 and 29 of Elesar by myself.

Tools: NCBI, PhagesDB, DNAMaster

Procedure:

  1. Opened the saved file from the previous lab, which had already be auto-annotated and had the template loaded.
  2. Recorded the SSC, CP, SCS, GAP, and LO for gene 28.
  3. Blasted the product of the gene using both the PhagesDB and NCBI databases. Recorded results under Blast-Start.
  4. Highlighted ORF in the frame viewer and used a Kibler7 Scoring Matrix and a Karlin Medium Spacing Matrix to find the Z score and Final Score.
  5. Repeated steps 2-4 for gene 29.
  6. Posted and saved file.

Results:

Blast results for Gene 28

Blast Results for Gene 29  (Blast results were the same for NCBI and PhagesDB so only NCBI shown)

Conclusions and Future Work: I was able to succeed in annotating the gene by myself. I noticed the importance of blasting using both databases as they could have different results such as in the case of Gene 28. In the future I will continue working on annotating Elesar as well as filling out the rest of the required parts of the annotation.

Category: Sriram Avirneni | LEAVE A COMMENT
January 30

BLAST Basics

Purpose and Rationale: Continue learning basics of phage annotation by comparing genes of Elesar to those found in BLAST results.

Procedure:

  • Opened Phage Elesar in DNA Master, using saved dnam5 file
  • Blasted Elesar Gene 1 in phagesdb.org and learned basics on comparing BLAST results to findings
  • Found gene 1 to not have any matches in phagesdb.org

  • BLASTed Gene 1 in the NCBI page, but still had no match
  • Following instructions from the SEA PHAGE Annotation Key, added to the gene’s notes that there are no significant BLAST alignments.
  • Continued reviewing the 15 Guidelines for Phage Annotation with the class and adding small notes in the same box as previous.                                                                                                    
  • Worked to find the correct start to Gene 1 by comparing start codons, coding potential, and again BLASTing the potential new gene in phagesdb and NCBI. 
  • Saved and closed the project.

Results: We have changed the location of the start of gene 1, and learned how to compare BLAST results to the query.

Future Plans: Continue annotating the genome of Elesar. Refine skills and learn how to use a computer.

Category: Uncategorized | LEAVE A COMMENT