January 29

Elsar BLAST Practice 1/28/19

Elsar BLAST Practice 1/28/19

Rationale: Learn basic annotation tools, and become familiar with BLAST NCBI database.

Procedure:  

  • DNA Master was opened
  • Elsar FASTA file opened and auto annotated
  • The reading frames were examined and adjusted
  • Genes 1 and 2 were selected to BLAST
  • Results from BLAST were examined and used to start annotation
  • Gene 14 of Elsar was chosen at random to BLAST-Start the gene in the notes
  • the results of Gene 1 and 14 were posted on DNA Master

The procedure of the bLAST was used as a practice BLAST to familiarize with the NCBI BLAST results. These results allow one to compare the query DNA sequence to similar sequences of DNA, and potentially allows identification of conserved DNA in multiple genome.

Gene 14

SSC: CP: SCS: ST: BLAST-Start: hypothetical protein [Arthrobacter sp. ok362], gene 14, NCBI, Q :S, coverage, 5e-58 Gap: LO: RBS: F: SIF: SIF-Syn

Results: 

Conclusions: The video played in the lab helped a great deal on understanding the results of a BLAST gene. With the results of the BLAST, BLAST-Start was done on gene 14. The results from BLAST allow one to compare the query DNA sequence to similar sequences of DNA as one means of verifying one gene or multiple genes. This helps researchers understand the function of the gene and each protein.

Future plans: Learn the basics of annotation through the SEA: PHAGES website and from the lab TAs. BLAST all of the genes of Napoleon B and compare the results of the BLAST with different databases such as Genemark and the auto-annotation results.

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
January 29

BLAST Practice 1/28/2019

BLAST Practice 1/28/2019

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
  2. A FASTA file was opened (File > Open> FastA Multiple Sequence File)
  3. The contents of the file were exported (Export > Create Sequence from this Entry Only)
  4. The contents of the file were Auto Annotated (Genome > Auto Annotate > click “yes”)
  5. The reading frames were examined (Genome > Frames> click “ORFS” button)
  6. Genes 1 and 2 were selected to BLAST (click on gene number > BLAST tab > Qblast against public database via NCBI server > Blast this gene)
  7. Results from the BLAST were examined and used to start filling in the annotation notes

Results

The procedure detailed above was simply practice that yielded the above results. These results show what an auto-annotated genome would look like after being BLAST-ed before the annotation notes have been updated. These results allow one to asses whether or not a gene that was marked as a gene during auto-annotation is actually likely to be a gene and it gives a frame of reference for comparing the query DNA sequence to other DNA sequences.

Conclusion

The results of the practice BLAST can be seen above. These results allow one to compare the query DNA sequence to similar sequences of DNA as one means of verifying that a gene is a real gene, and as a way to help understand the function of the gene and potentially identify areas of conserved DNA in multiple genomes.

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.

Category: Lucy FIsher | LEAVE A COMMENT
January 29

1/28 ~ BLASTING Basics and Practice

Rationale: Practice BLASTING on the PhagesDB (Protein) Server as well as the NCBI (Protein) Database.

 

Materials:

  • Computer
  • DNA Mastering Application
  • NCBI Database (Protein)
  • PhagesDB Database (Protein)
  • Annotation Key
  • GeneMark

 

Procedure:

  • Opened DNA Mastering on the computer and opened Elesar file
  • Auto-annotated and opened up frames view of the genes
  • Opened up PhagesDB and NCBI Databases on separate tabs
  • Copied the protein sequence from the genes (This case Gene 1) and inputted into the BLAST in both NCBI and PhagesDB
  • Recorded the annotations needed (SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn)

Observations:

  • This screenshot shows the annotations in the notes box for the gene. Not all annotations are filled in because time ran out

 

Conclusion/Next Steps:

  • Learned more about DNA Mastering and the process needed to completely annotate and document the gene. With this new information, will be able to apply to the annotation of NapoleonB
Category: Justin Yu | LEAVE A COMMENT
January 28

January 28 2019- BLAST and Annotations

Purpose: The purpose of this lab was to learn how to do a BLAST on NCBI and PhagedDB, and to learn how to annotate a gene.

Tools/Procedures: 

Tools:

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

Procedures:

  1. A BLAST was performed on Elesar gene 1 with NCBI and PhagesDB.
  2. The start was found to be at bp 45, making the coordinates of the gene 45, 353.
  3. The Coding Potential (CP) was found to include all coding potential identified by GeneMark.
  4. The start choices predicted by Glimmer and GeneMark were the same, but neither was used.
  5. The BLAST results found no significant alignments for Elesar 1.
  6. The annotation was saved.

Results:

The BLAST results yielded no significant alignments for Elesar 1. The annotation was started but was not completed.

Conclusions:
Because the E value for Elesar gene 1 was so high, ranging from 3.4 to 10, it was concluded that there was no significant alignment found. Both NCBI and PhagesDB did not have a gene that significantly matched Elesar gene 1. The annotations for this gene were begun, but not finished. The start of the gene was moved to the longest open reading frame, which also included all of the coding potential identified in GeneMark. While the start codon was TTG and this is rare, it is possible, and this start was identified as an overall better fit.

 

Future Work: 
Future work will include finishing the annotation of Elesar gene 1, and then proceeding to BLAST more of Elesar’s genes. Further practice will be done with Elesar to later annotate NapoleonB.

Category: Lucy Potts | LEAVE A COMMENT
January 28

1.28.19 BLAST Analysis

1.28.19 BLAST Analysis

Rationale: The next step in learning how to accurately and scientifically annotate a genome was considered to be learning BLAST analysis techniques. Therefore, this laboratory session was dedicated to practicing BLAST analysis using the Elesar phage genome.

Procedure:

  • Opened DNA Master
  • Opened FASTA File Elesar
  • Export –> Create Sequence from this entry only
  • Genome –> Auto Annotate –> Annotate
  • Copied Gene 5 protein sequence and inputted to NCBI BLAST database
  • Analyzed Results

Results:

[Elesar, 5, NCBI, Query 1 to Subject 1, 99%, 0.0, terminase large subunit]

Conclusions: Since the query had a 99% coverage and an e value of 0.0, it was concluded that Gene 5 of Elesar was likely coding for the terminase large subunit. This is backed by BLAST evidence, as 97% of the amino acids were a direct match to the NCBI sequence and 98% of the amino acids could be considered positive.

Future Plans: In the next lab session, the full annotation process will be assembled and put together. At this point, it will be possible to practice annotations on Elesar, and finally later on NapoleonB.

Category: Henry Burns | LEAVE A COMMENT
January 28

Lab Day 3: Blast intro

Rationale

In the beginning of the lab, we learned about lambda phage cycle. We also learned about the purpose of Blast, how to perform Blast, and how to read the results from the NCBI database. Learned how to make the LORF (longest open reading frame), and update SSC, CP, SCS, and the rest of the template

Observations

Next steps

Make LORF for other genes and update template (SSC, CP, SCS, etc) for that gene.

Category: Soo-Un Jeong | LEAVE A COMMENT
January 28

DNA Day 3

28 January 2019 ✷ DNA Master + BLAST

Rationale: DNA from Elesar gene 1 was BLASTed in order to see if other genes exist in the NCBI database.

Procedure

  • The FASTa file for Elesar was opened and autoannotated.
  • The protein sequence for gene 1 was opened and copied and pasted into the BLASTp tool on NCBI’s website and run to find matching sequences,

Results

Conclusion

The NCBI BLASTp result was not very effective in locating a similar gene for Elesar Gene 1. This just means that similar genes are not stored in the database as of yet. The E value is really high for each of the results and this means that it is very likely the results are random. The identity percentages are low as well and thus the protein sequences are not very closely related.

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 28

BLAST Practice 1.28.19

Rationale:

To continue practice annotation of Elesar by becoming more familiar with how to BLAST.

Procedure:

  1. Using an already open and auto-annotated Elesar Mastering DNA window, a BLAST search of gene 1 was conducted using the in program BLAST search feature.
  2. A BLAST protein search for gene 1’s protein sequence was also conducted using the NCBI BLAST program.
  3. Step 2 was repeated for the PhagesDB BLAST program.
  4. Analyzed Results.

Results:

For both NCBI and the DNA Master BLAST searches, all hits had scores less than 40. For the Phages DB BLAST there was a hit with a score of 178 and an E value of 2e-45, with the hit being the Elesar gene 1 unknown protein.

Conclusions:

Based upon the results it can be concluded that the protein for gene 1 is unknown as shown by the BLAST result from Phages DB, considering that both the sequence blasted and the hit were actually the same genes from Elesar.

Next Steps:

I will continue practicing genome annotation via DNA Master using the phage Elesar’s genome in order to eventually annotate the genome of the phage Napoleon B.

Category: Nathan Newton | LEAVE A COMMENT
January 28

Beginning Annotating Elesar Gene 1 1/28/19

Rationale: In order to learn how to annotate a genome I practiced annotating the first gene of Elesar.

Tools: NCBI BLAST, DNAMaster

Procedure:

  1. Began by auto-annotating the genome of Elesar using the template set-up from last lab.
  2. Opened the frames view and looked at the open reading frame of gene 1. Due to there being a larger Open Reading Frame(ORF) possible I moved the ORF to the Largest Open Reading Frame(LORF).
  3. Noted in the annotation the Start and Stop Coordinates(SSC), the Coding Potential(CP), and Start Choice Source(SCS).
  4. Blasted the product using the NCBI database, which turned up no good matches, which I noted under Blast-Start.
  5. Noted the fact that this is the first gene for GAP, and noted that this is indeed the LORF.
  6. Opened up frames and highlighted the ORF and clicked RBS. Using a Kibler7 Scoring Matrix and a Karlin Medium Spacing Matrix, I recorded the Z Score, Final Score, and whether or not it was the best Final Score.
  7. Posted and saved file.

Results:

Results of RBS.

Conclusion and Future Work: I’ve learned the first few steps to annotating a genome. Now I will begin working on annotating genes by myself.

Category: Sriram Avirneni | LEAVE A COMMENT
January 27

Beginning Auto-Annotation of Phage Elesar

Shepard Saabye

January 23rd, 2019

 

Purpose and Rationale: Begin learning the basics of DNA Master by annotating Phage Elesar.

Procedure:

  • Implemented a new code to replace an outdated procedure inside the local settings screen.

 

  • Reloaded Elesar and DNA Master, and initialized auto-annotation.
  • Opened frames, and found all open reading frames within the genome.

  • Due to complications with computers, this was as far as the class got during lab time. Proceded to clos programs, and followed instructions for QTM.

Results: The genetic makeup of Phage Elesar contains 66 potential genes, of which only 11 are reverse genes. The genome is nearly 43 thousand base pairs long.

Future Steps: The auto-annotated genome of Elesar is ready to be analyzed BLASTed in a separate program based on an online platform, from Genemark. With that, we will be able to potentially find the lineage, and functions of the genes.

Category: Uncategorized | LEAVE A COMMENT