January 25

Annotation of Elesar

1/23/19

Rational:

To further annotate the genome of Elesar and starting to check for mistakes in the auto-annotation as well as learn how to calculate gaps and overlaps between genes.

Procedure:

  • Clicked preferences under files
  • Clicked local setting and new features
  • Added SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn into the template notes box
  • Calculatted the gap and overlap between some example genes on the questions that matter worksheet
  • Calculated the gap by finding the difference between the end of one gene and the start of the next minus one
  • Calculated the overlap by finding the difference between the end of one gene and the start of the next plus one

Observations:

  • Some of the foward and reverse genes overlap or are too close to be correct (22/24, 24/25, 32/33, and 55/56 are examples of this)

Results:

Some of the genes like 22 (which is reverse) and 24 (forward) are too close and indicate a possible mistake in the auto-annotation. Next lab I will calculate the gap and overlap between genes (especially between forward and reverse genes).

January 25

Further Annotation of Phage Elesar (1/23/19)

Rationale:

The purpose of this lab was to further understand DNA Master and the information presented for the future.

Tools:

  • Computational Device
  • DNA Master program
  • Elesar FASTA File

Results:

  • After the presentation about Ebola was given, DNA Master has been opened and the phage Elesar FASTA file was uploaded.
  • The file was then exported in order for the sequence to be created.
  • After it was exported, it was auto-annotated from the option of genome then auto-annotate.
  • After annotation, the information under Features was explained such as gap and overlap calculations and practice was done with gene 19.
  • Then, the frames were explained and purpose for their different structures along with the purpose of six reading frames.
  • The ORF’s were viewed and future tools were discussed.

 

Conclusion:

From this lab, DNA Master was explained into more detail such as which preferences were needed and which steps needed to be taken to view the open reading frames. Future plans were explained and which tools were needed to accomplish those tasks. We also practiced gap and overlap calculations in order for future research.  Along with the calculations, the 5′ to 3′ for each base pair was explained and they were supposed to be read.

 

Future Work:

In the next lab, DNA Master will further be explained and actual annotation will begin.

 

January 25

January 25th labs

  • JANUARY 25th, 2018
  • OBJECTIVE:
    • To auto-annotate the genome of Elesar, and learn how to calculate gaps and overlaps within the genome
  • PROCEDURE: 
    • DNA master was downloaded 
    • And a word document was copied and pasted into DNA master for annotation settings
  • RESULTS: 
    • Error code kept occurring when attempting to auto-annotate Elesar’s genome
  • CONCLUSION: 
    • Annotation settings were successful updated
  • FUTURE STEPS: 
    • Work on finding reason why DNA master will not auto-annotate
January 25

1-23-19 — Gene Annotation Intro and Template Setting

Gene Annotation Intro and Template Setting

Date: 1-23-19

  • Rational
    • The rational for this lab was to add a template to DNAMaster for autoannotation and understand the counting and significance of gaps and overlaps between gene sequences.
  • Procedure
    1. DNA Master was opened.
    2. A template was added by going to File > Preferences > Local Settings > New Feature. ‘Insert template into notes during autoannotation’ was selected and a template code was pasted in the text box.
    3. The code “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn” was added with the following meanings:
      • SSC: Start/Stop Coordinates
      • CP: Coding Potential
      • SCS: Start choice source
      • ST: Starterator
      • BLAST-Start: The best BLAST match for the gene
      • Gap: Gaps or overlaps
      • LO: Longest ORF, for genes with ~10 bp between start codon and upstream stop codon
      • RBS: Ribosome binding sites
      • F: Gene function
      • SIF-BLST,-HHPred,-Syn: Supporting information for the function
    4. Elesar was opened and autoannotated with this template saved.
  • Results
    • The template appeared in the notes section of the autoannotated Elesar.
  • Conclusion
    • Using the template allows for more accurate presentation of annotation information in the notes section.
  • Future Plans
    • This template will be used for future annotations. As gene annotation experience is gained, it will be easier to recognize and analyze trends as well as correctly manually annotate gene sequences.
January 25

January 25, 2019 The Forgotten Cure 1

The Forgotten Cure by Anna Kuchment is a fascinating book. While I knew who discovered bacteriophages, I did not know how they came across them or how deep their research went. Felix d’Herelle not only led an interesting life but proved to be a great scientist. It is interesting that he never attended higher education, but loved science so much he studied it on his own. D’Herelle’s interest in phage led him to not only discover their existence, but he was able to learn how bacteriophage entered bacteria and destroyed them with simple observations and no electron microscope.

D’Herelle’s work with locusts led to his discovery of bacteriophages, but without his work with a dysentery outbreak, he might not have carried his work as far as he did. It is amazing to me to think of how phages were already working in patients with dysentery to keep the bacterial colonies in check before we even knew phage existed. D’Herelle’s close observation of his patients shows his dedication and interest in his work.

With so many major breakthroughs in science around the time bacteriophage were discovered, it is not shocking to me that phage therapy failed to spread at this time. Without being able to see phages, it was harder for scientists to study their structure and how they worked. Regulation of clinical trials and research was also a major reason phage therapy failed to spread. This combined with little government funding persuaded many scientists to move on to other areas of research. When d’Herelle first published about phages, there was no television or radio, which also slowed the spread of phage therapy in his time. Also, because he was self-taught, many scientists were reluctant to trust him at first.

Max Delbruck and Salvador Luria and the Phage Group made many more discoveries with the help of bacteriophage. Their work with phages led to Hershey and Chase’s experiment and the discovery of DNA as the genetic material. They also learned how phages attack and kill bacteria. While they worked on phages, the scientists limited the number of phages they studied to seven, limiting their impact on areas such as the diversity of phages.

Reading the first few chapters of The Forgotten Cure has introduced me to many new topics in phage discovery and therapy. I am excited to continue reading and learn more about bacteriophages.

January 25

Annotation Practice on DNA Master 1/23/19

Title: Annotation Practice on DNA Master

Date: 23 January 2019

Rationale: In this lab, the goal was to practice auto-annotation and experiment with some of the finer details regarding ORFs and gene overlap/gap. This lab also showed where the computer leaves ambiguity and how a gene must also be manually annotated.

Tools:

  • Microsoft Surface Pro 5 Tablet
  • DNA Master Software

Procedure: The FASTA file for phage Elesar was uploaded to the DNA Master software, exported, and “auto-annotated” in order to visualize individual ORFs and tRNAs. Once the Features list was visible, the Frames window was obtained by selecting the “Frames” option from the “DNA” drop down list. An annotation template was also inserted into the “Preferences.”

Results/Observations: The auto-annotated results showed the potential start and stop locations of potential genes (ORFs), a picture of the annotation is shown below (green values are forward reading frames, red values are reverse reading frames):

Out of 66 total found ORFs (and 1 tRNA), 11 were determined to be reverse reading frames while the other 55 were forward reading frames. Many ORFs were also observed to have gaps or overlap based on the start and stop location.

Conclusions/Next Steps: The gaps in ORFs are due to promoter regions in between each gene (instead of them being “backed up” against one another) while overlaps can be due to ambiguity in the computer’s annotation, which must be determined manually. The next steps for this lab are to start BLAST-ing the observed genes and comparing them (as proteins) against known sequences and functions. Also, the overlaps and gaps will have to later be accounted for manually.

 

January 25

1/23/19 Annotation Intro

Title: 1/23/19 Annotation Intro

Rationale: 

The purpose of today’s lab was to be introduced to the process of gene annotation, finalize the set preferences for DNA master, and learn about analyzing information given in auto annotation with phage Elesar.

Tools: 

  • DNA Master Auto Annotate Function
  • FASTA file “Elesar”

Procedure:

  • Opened DNA master and uploaded template “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn” to begin auto annotating Elesar.
  • After uploading the template, the genome was auto annotated.
  • The ORFs of the genome were examined and identified by using DNA –> Frames –> ORFs

Results

  • The majority of the genome contains forward ORFs, with gene number 19 being among some of the longest.
  • Auto annotation only revealed approximately 11 reverse ORFs, making up only 16.67% of the genome.

Conclusions:

The genome of Elesar’s lack of reverse ORFs can also indicate a lack of primers that begin coding in the reverse direction. Also, the size of the ORFs, regardless of direction, could have a correlation between the size of proteins produced and/or the number of proteins produced within that gene.

Next Steps:

Need to continue with the familiarization of DNA master so it can be fully utilized to manually annotate genomes. Further practice is needed to begin manually annotating NapoleonB and discovering which proteins are coded within the genome.

 

January 25

Elesar Annotation 1/23/19

Rationale

Today we will learn more about reading frames and explore more of DNA Master using the genome of Elesar.

 

Procedure

  • DNA Master was opened and the template “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST:SIF-HHPred: SIF-Syn” was uploaded for future annotations.
  • Elesar was opened using the FastA file and the new template was employed to autoannotate.
  • ORFs were also accessed to be observed and begin the process of learning their functions.

 

Results

More information on gaps and overlaps, as well as ORFs were presented. Knowledge about DNA Master and ORFs are still limited, therefore no notable results were achieved from the day. The new template may be used for future autoannotations.  A better understanding of DNA Master and the different tools in the program was reached instead.

 

Conclusion/Future Steps

The tools being used in DNA Master will allow for the autoannotation of NapoleonB in the future. DNA Master will be explored more using Elesar to become familiar with the program.

 

January 25

DNA Master Annotation Practice 1/23/2019

DNA Master Annotation Practice 1/23/2019

Rationale

The rationale behind these procedures is to learn how to annotate genes in DNA master while also adding several features to the auto-annotation sequence.

Tools/Procedure

  1. Open DNA Master
  2. Open File> Preferences > Local Settings > New Feature > add the following code:
    SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST:
    SIF-HHPred: SIF-Syn
  3. Open a FASTA file (File > Open> FastA Multiple Sequence File)
  4. Export contents of the file (Export . Create Sequence from this Entry Only)
  5. Auto Annotate contents of the file (Genome > Auto Annotate > click “yes”)
  6. Examine reading frames (Genome > Frames> click “ORFS” button)
  7. Compare gene locations paying attention to gaps and overlap

Results

The procedure detailed above was simply practice that yielded the above results. These results show what an auto-annotated genome would look like for future reference in later annotations. They also show the 6 possible reading frames on which genes can be found. The genes shown highlighted in green signify genes read in the forward direction while genes highlighted in red show reverse genes. These results allow me to compare gaps and overlaps between genes to determine in the auto-annotation was accurate in determining genes in the genome.

Conclusion

The results of the practice auto-annotation can be seen above. An annotated genome that can be reviewed by a human. By examining the reading frames one can determine if the genes marked in the auto-annotation are likely to be legitimate coding strands for genes or not. While there were no tough calls to make in today’s lab, genes 25 and 22 look unlikely to be legitimate and this will need to be further examined.

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 also likey study genes 25 and 22 to ensure that my initial reaction to their legitimacy is accurate.

January 25

DNA Master Test Run & Gene gaps/overlaps 1.23.19

Rationale:

To begin the genome analysis of phage NapoleonB, DNA Master has been chosen to be the software to annotate the genetic sequence. Phage Elesar was picked to be the sequence for the tutorial, in order to familiarize with the functions and features of the software.

Materials:
  • Laptop
  • DNA Mastering Program
  • Gene sequence of phage Elesar
Procedure:

Genome -> auto-annotate -> annotate.

Gene auto-annotation function in DNA Master was explored, the potential genes in the whole sequence were found, and from the results we can calculate the gaps/overlaps of the genes.

If gaps -> subtract and minus one.

If overlaps -> subtract and add one.

Results/Next Steps:

DNA Master is now operational on the personal laptop so the next lab would be learning how to BLAST the genes auto annotated from phage Elesar for testing.