January 31

Elesar Gene Annotations 1/30/19

Rationale: Annotated genes 6 and 7 of elesar

  1. Gene 6
    SSC: 3685-4974
    CP: yes
    SCS: both
    ST: NA BLAST-Start: matches with Nandita, 7, phagesdb, q1:s1, 97%, 0.0
    Gap: 16
    LO: yes
    RBS: Kibler7, Karlin Medium, 3.215, -2.059, yes
    F: portal protein SIF-BLAST: portal protein, phagesdb, Nandita_7, MH834621, 97%, 0.0
    SIF-HHPred:
    SIF-Syn

  2. Gene 7
    SSC: 5025-6221
    CP: yes
    SCS: neither
    ST: NA
    BLAST-Start: matches with Nandita, 8, phagesdb, q1:s14, 93%, 0.0
    Gap: 50
    LO: no
    RBS: Kibler7, Karlin Medium, 1.823, -6.049, no
    F: capsid maturation protease
    SIF-BLAST: capsid maturation protease, phagesdb, Nandita_8, MH834621, 93%, 0.0
    SIF-HHPred:
    SIF-Syn

Results/Conclusion:

Annotated 2 genes. Gene 6 I determined to be an RBS because it had the best values for Z score and F score. I changed the starting codon on gene 7 based on the ORF map suggested value and concluded that it was not an RBS.

Future Steps: Figure out if my annotations were correct and figure out how to successfully use template notes.

Category: Rachel Melone | LEAVE A COMMENT
January 31

Elesar BLAST 1/28/2019

Rationale: BLAST elesar fasta file

Process

  1. auto-annotated again
    1. genome -> auto-annotate -> annotate
    2. auto-annotate with pecaan
      1. entered phage name “elesar”
      2. uploaded fasta file
      3. clicked “process”
    3. copied pecaan documentation, pasted it into documentation tab of dna master
    4. removed documentation previously there
    5. clicked “parse”, once directed to a new window, clicked the new “parse” button.
  2. Viewed ORFS
    1. DNA -> frames
      1. ORFS button in right hand corner
  3. Learned how to change start codon
    1. changed start codon of gene 1 from 84 to 45
  4. BLAST gene with DNA Master
    1. select gene
      1. blast tab
      2. blast this gene
      3. wait for BLAST
      4. save to database tab
        1. select E value
        2. save to database

Results/Conclusion:

I could view the different ORFS and evaluate the best starting codon.

Future Steps: learn how annotate a gene

Category: Rachel Melone, Uncategorized | LEAVE A COMMENT
January 31

Gene 32 and Gene 41 Annotation

Gene 32 and Gene 41 Annotation

Rationale: Finish the annotations of Elesar gene 1 and begin Annotations for gene 32 and gene 41

Procedure:

  • DNA Master was opened and Elesar FASTA file opened
  • Reading frames were opened and genes 32 and 41 were observed
  • Both genes were blasted through NCBI Protein Database
  • Elesar gene was pulled up on Genemark
  • SSC: CP: SCS: BLAST-Start: Gap: LO: RBS were the categories that were annotated

Results:

Blast results shown above for gene 32 and 41 are both from the NCBI Protein database. The images above also show results for each query of the two genes.

Gene 32

SSC: 23223 26093 CP: yes SCS: Both-Both Blast-Start: hypothetical protein PBI_BRIDGETTE, 30, NCBI, Q601: S62, 81%, 0.0 Gap: 7 LO: yes RBS: Kibler 7, Karlin Medium, 2.117, 4.852, no

 

Gene 41

SSC: 31001 31219 CP: yes SCS: Both-Both Blast-Start: Mycobacterium virus Brujita, 35, NCBI, Q7: S2, 60%, 4e-13 Gap: 607 LO: yes RBS: Kibler 7, Karlin Medium, -6.128, -8.014, no

Conclusions:

Annotations of genes 32 and 41 were not bad after the TA of the class showed the class what each letter meant under the annotation key. Observing the blast was easy while observing genemark results were hard, but with the help of the lab TA’s, it was possible.

Next Steps:

Start annotations for NapoleonB.

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
January 31

Annotation of Elesar 1/30/19

Rationale

Today we will finish annotating Gene 1 of Elesar and will independently annotate other genes in the genome.

Procedure

  • The work done on the annotation of Gene 1 was completed by re-opening the FASTA file and annotations.
  • Genes 42 and 43 were assigned to annotate for practice, and the following categories were completed with respect to the genes assigned: SSC:, CP:, SCS:, ST:,BLAST-Start:, Gap: LO:, RBS:

Results

Blast results are shown above for Gene 42 and 43. The results show the NCBI database and the matching results for each query.

Gene 42: SSC: 31255, 31935, CP: yes, SCS: both-cs, BLAST-Start: [hypothetical protein PBI_MAJA_36, 36, NCBI, 46, 2, 2e-46], Gap: 3 overlap, LO: N/A, RBS: [Kibler 7, Karlin Medium, 2.325, -3.883, no]

Gene 43: SSC: 31932, 32198, CP: yes, SCS: Glimmer, BLAST-Start: [hypothetical protein PBI_RYAN_46, 46, NCBI, 1, 1, 2e-53], Gap: 3 overlap, LO: N/A, RBS: [Kibler 7, Karlin Medium, 2.214, -4.470, no]

Conclusion/Next Steps

The practice of annotating the genes can be beneficial for future use. Annotations will occur with NapoleonB and it is imperative that an understanding DNA Master is possessed.

Category: Emily Gaw | LEAVE A COMMENT
January 31

Basics of Blast 1/28/19

Rationale

Today we will learn more about the BLAST feature in DNA Master by BLASTing genes in the Elesar genome.

 

Procedure

  • Elesar was opened again in DNA Master and the FASTA file was autoannotated.
  • Gene 1 of Elesar was auto-annotated using the details from the annotation key and auto-annotation page.
  • The protein sequence of Gene 1 was copied and inserted into the protein BLAST at NCBI.
  • The BLAST results were then interpreted.

 

Results

Blast results are inserted above for Gene 1. Hypothetical protein A of Arthrobacter sp Hiyo is shown to have the highest score. A gap of two is indicated as well.

 

Conclusions/Next Steps 

A portion of the annotation of Gene 1 was done as a group and will be finished by the next lab. The Gene annotation of Elesar will continue until a better understanding of DNA Master and Blast is reached.

Category: Emily Gaw | LEAVE A COMMENT
January 31

BLAST Practice and Annotation Notes 1/30/2019

BLAST Practice and Annotation Notes 1/30/2019

Rationale

The rationale behind these procedures is to learn how to annotate genes in DNA master by looking at BLAST results and using them to add information to the annotation notes.

Tools/Procedure

  1. DNA Master was opened and the previous Elsar file was loaded
  2. The annotation notes for the following fields – SSC: CP: SCS: BLAST-Start: Gap: LO: RBS: – were completed for gene 1
  3. In order to complete the annotation notes the start codon number for gene one was adjusted from 84 to 45
  4. Genes 10 and 11 were BLAST-ed and the annotation notes for the following fields – SSC: CP: SCS: BLAST-Start: Gap: LO: RBS: – were completed

Results

Gene 1 – SSC: 45,353 CP: yes SCS: both-cs BLAST-Start: no significant BLAST alignments Gap: first LO: yes RBS: Kibler7, Karlin Medium, 1.222, -6.751, no

Gene 10 – SSC:7514,7813 CP: yes SCS: both BLAST-Start: no significant BLAST alignments Gap: 15 bp LO: yes RBS: Kibler7, Karlin Medium, 2.299, -4.080, yes

Gene 11 – SSC: 7829, 8230 CP: Yes SCS: Both BLAST-Start: Aligns with Arthrobacter phage Ryan, NA, NCBI, q1:s1, 95%, 2e-70 Gap: 15 LO: yes RBS: Kibler7, Karlin Medium, 2.565, -3.515, no

Conclusion

These results show the completed structural annotation notes for genes 1, 10, and 11. Of these genes, only gene 11 has significant BLAST results, meaning that gene 11 has a sequence of DNA very similar to other DNA found in bacteriophage. Genes 1 and 10, however, do not have significant BLAST results and therefore are not highly conserved among known genomes. This could mean that hypothetical genes 1 and 10 are not real genes, or it could mean that they are unique and could code for more unique protiens.

Future plans

In the future, I will use what I learned how to do in this procedure when I am analyzing the genome from Napoleon B. I will perform auto-annotations as well as many other forms of testing on that genome in the lab periods to come. I will likely BLAST all of the genes in Napoleon B and compare those results to genemark and the auto-annotation results in order to asses that all genes are in fact genes and to fully annotate the genome. I will also be sure to fill in all of the basic information that is required in the annotation notes to make scientific inquiry easier in the future.

Category: Lucy FIsher | LEAVE A COMMENT
January 31

DNA Day 4

30 January 2019 ✷ DNA Master + BLAST

Rationale: DNA from Elesar gene 1 was annotated.

Procedure

  • The FASTa file for Elesar was opened and auto-annotated.
  • The “features” tab was opened and within the “notes” box, the code for genes 2 and 3 were annotated.
  • The “frame” was pulled up and the ORF was viewed to see if it could be elongated. The start and stop coordinates and gap/overlap were looked at.
  • The genes were each blasted with phagesdb in order to find a similar gene. This information was included in the annotation.

 

Results

the annotations for each gene are as follows:

Gene 2

SSC:[346,690] CP:[yes] SCS:[both] ST: BLAST-Start:[Andrew, 1, phagesdb, 1:1, 62%, 3e-17 ] Gap:[8bp overlap] LO:[no] RBS:[Kibler7, Karlin Meduim, 3.167, -2.177, no]

Gene 3

SSC:[954,1352] CP:[yes] SCS:[both] ST: BLAST-Start:[Ryan_4, phagesdb, 1:1, 6e-70, 100%] Gap:[263bp gap] LO:[yes] RBS:[Kibler7, Karlin Medium, 2.573, -3.709, no]

Conclusion

Annotation is useful in research in comparing different genes within differing species that potentially contain the same gene. Practicing this specific skill will be important because the process was somewhat confusing, but it is surely an important method for genetic research.

Future plans

This same method will be applied to Napoleon B, a phage isolated in 2018, in order to analyze its genome.

Category: Lily Goodman | LEAVE A COMMENT
January 31

01/30/19- gene annotation of gene 14 and 15

01/30/19

Rationale:

to learn genetic annotation on DNA master.

Procedure:

  1. open  DNA master
  2. Open FastA file for elesar using the following steps, File>open>FastA multiple sequence file> Choose Elesar.FastA
  3. click export on window with the extracted file and then choose Create sequence from this entry only.
  4.  to see the frames, go to DNA> Frames.
  5. then click ORF on the new window to see the highlighted genes.
  6. Click on the BLAST tab and then choose BLAST this gene.
  7. zoom in on gene one in the frames window and highlight gene length so that it is as long as possible.
  8. lengthen the gene length by changing the starting base pair number of the gene.
  9. press the calculator icon and then click post.
  10. Using the annotation key and the BLAST results, finish the description of gene 1 as the template requires.
  11. repeat the same steps for gene 14 and 15.
  12. click post to save changes every time data is altered.
  13. save the file.

Results:

Gene 1:SSC:45-353 CP: yes SCS:Both-CS BLAST-Start:no significant BLAST alignment Gap: first gene LO:yes RBS:Kibler7,Karlin medium,1.222,-6.751,no

Gene 14:SSC:8837-9250 CP: yes SCS: both BLAST-Start: Arthrobacter sp. ok362, na, NCBI, Query 14 to subject 1,100%, 0.0E0 Gap:4 LO: yes RBS: Kibler7, Karlin Medium,2.801, -3.226, no

Gene 15:SSC:9307-9969 CP: yes SCS: both BLAST-Start: Arthrobacter phage sp. Hiyo6, 05270,NCBI, Query15 to Subject1,100%,4e-97 Gap:56 LO: yes RBS: Kipler7, Karlin medium,2.801, -3.226, no

Conclusion:

None of the above genes 14 and 15 had significant matches but did not have low  final scores. These two genes have matches with a close to 0 e value.

Future Steps:

continue practice with DNA Master and gene annotation.

 

Category: Uncategorized | LEAVE A COMMENT
January 31

01/28/19- BLAST and annotation of gene 1

01/28/19

Rationale:

to begin learning how to annotate a genome

Procedure:

  1. open  DNA master
  2. check the “insert template into notes during Auto Annotation” box. Then Apply>OK
  3. Open FastA file for elesar using the following steps, File>open>FastA multiple sequence file> Choose Elesar.FastA
  4. click export on window with the extracted file and then choose Create sequence from this entry only.
  5.  to see the frames, go to DNA> Frames.
  6. then click ORF on the new window to see the highlighted genes.
  7. Click on the BLAST tab and then choose BLAST this gene.
  8. zoom in on gene one in the frames window and highlight gene length so that it is as long as possible.
  9. lengthen the gene length by changing the starting base pair number of the gene.
  10. press the calculator icon and then click post.
  11. Using the annotation key and the BLAST results, fill in the description of the gene for start/stop coordinates and coding potential.
  12. click post to save changes.
  13. save the file.

Results:

the gene annotation of gene 1 yielded the following: SSC:[45-353]CP: yes SCS:Both-CS

Conclusion:

the BLAST results yielded no significant gene alignment. there is currently no determinable function of this gene.

Future Steps:

complete the annotation of gene 1 and continue annotation.

Category: Uncategorized | LEAVE A COMMENT
January 31

1/30/19 Annotation Practice with Phage Elesar

Rationale: 

The purpose of today’s lab was to annotate given genes for phage Elesar.

Tools:

  • Laptop
  • DNA Master
  • Genemark
  • NCBI BLASt

Procedure:

  • Continued with annotation of gene one from 1/28/19.
  • Determined preferred RBS for gene one would make gene way too small, so disregarded the suggested RBS as the z score was not good enough and changing the start would make the gene too small.
  • Was assigned genes 30 and 31 to annotate.

Results: 

  • Annotation Notes For Gene 30:
    • SSC:22118, 22477 CP:yes SCS:Both-Both BLAST-Start: Nandita gp29, NCBI,  q1:s1, 78%, 9E-62 Gap:overlap 1bp LO: yes RBS: Kibler7, Karlin Medium, 2.408, -3.770, yes
    • Protein BLAST result matched with a hypothetical protein produced by gene 29 of Arthrobacterphage Nandita with 78% coverage and an e score of 9e-62.
  • Annotation Notes for Gene 31:
    • SSC:22487, 23215 CP:yes SCS:Both-Both ST: BLAST-Start:Ryan, gp30, NCBI, q1:s1, 83%, 2e-139   Gap:9 LO:yes RBS: Kibler7, Karlin Medium, 2.117, -4.325, yes
    • Protein BLAST results yielded a hypothetical protein produced by gene 30 of Arthrobacterphage Ryan, with 78% coverage and an e score of 2e-139. In addition to this gene, the next best hit was with gene 30 of Arthrobacterphage Nandita, with 81% coverage but an e score of 4e-139.

Conclusions:

The two genes that were annotated both hit on the same phage, and this was the case with several other genes annotated by classmates nearby. It can be inferred that these different phages are in the same cluster, or share some similar trait that calls for this same hypothetical protein to be made. It is unaware of what the protein is and what the suggested function of the protein is as well. Both genes had excellent coding potential, and are very close in proximity to one another.

Next Steps:

It could be possible to annotate the surrounding genes for any known proteins. If they are discovered then at least a function of the hypothetical protein could be deducted from the surrounding genes. Further annotation practice using phage Elesar is required.

Category: Gabriel Andino, Uncategorized | LEAVE A COMMENT