Ashton Peckinpah

Tuesday, April 18, 2017

Objective: To finalize the data and research components of the project. Overall conclusions were drawn and written as a group. Then, the final poster was put together and creative components were made.

Methods:

1. Obtain Dot Plot from Lathan
2. Create results flow chart.
3. Draw overall conclusions.
4. Make final poster as a group.

In conclusion: A dot plot was created in order to assist in the comparison. The AM portion of the dot plot is noticeably lighter, meaning that there is less similarity. There is even more similarity within the AM sequence than with the AN or AK cluster.

Lastly, nucleotide sequence lengths were compared because we believed that varying lengths of the sequences could explain the extremely small overall value of similarity.

After comparing the data, the dot plot, and the sequence length, overall conclusions were drawn. The AM sequence was too short to be directly compared to the AN and the AK sequences. The AM sequence is so short that is has limited room to be similar to the other sequences. Moreover, the AN and AK sequences still very by about 500 base pairs, further explaining the difference in similarity. Ultimately, we concluded that tape measure proteins need to be more similar in length in order to complete and accurate comparisons.

Finalized Poster:

 

Ashton Peckinpah

Tuesday, April 11, 2017

Objective: To continue conducting our independent research projects: conducting research and gathering useable data. In order for this to be accomplished, a clearly defined research question was developed and a detailed outline for the project was developed. Additionally, a rough draft of our poster was completed and primary sources were found in order to support our research.

Methods:

1. Obtain all three nucleotide sequences for the alignment (one sequence from each cluster).
2. In a FASTA file, place each tape measure sequence following the correct format and name each sequence with an easily identifiable name.
3. Upload the FASTA file into Clustal Omega and run the alignment.
4. Analyze the results of the sequence alignment.

 

Tools Used: NCBI Blast, Clustal Omega, PubMed, and PhagesDB

In conclusion:

 

The AM sequence does matches far less with their AN or AK. Moreover, the AN and AK sequences have many more nucleotide base pairs in common. There are a few places where all three match; however, the overall results show very little similarity between the sequences.

Following this lab, we will use the data we conducted to create a dot plot that visually compares the three clusters. Moreover, we will future explore and try to understand why these clusters do not share a lot of similarities.

Ashton Peckinpah

Tuesday, March 28, 2017

Objective: To continue conducting our research and gathering useable data. A clearly defined research question was developed and a detailed outline for the project was developed.

Methods:

1. Identify two other clusters that have myovirdae morphology.
2. Identify two published phage genomes within each of those two clusters.
3. Identify the tape measure gene in each genome and obtain the nucleotide and protein sequences.
4. Run an NCBI Blast to observe the similarity between tape measure genes within the same cluster.

 

Tools Used: DNAMaster, Phamerator, and NCBI Blast.

In conclusion: We discovered that two clusters of myovirdae phages are within the AM and AK clusters. Moreover, the AM cluster only had one published genome; therefore, we could not use a NCBI Blast to determine the similarity between two tape measure genes within the cluster. This sequence will however be used when comparing the three clusters.

The two published phages that we used were Bennie and Greenhouse. After blasting the two phages tape measure proteins on NCBI Blast, we discovered that the two phages have a 93% nucleotide sequence similarity. Although this is lower than the 99% similarity between the two AN phages, it is still a very high match and is sufficient data.

After comparing the two AK phages, we were sure that tape measure proteins within a cluster share a lot of (over 90%) similarities in nucleotide sequence. After understanding this finding, we were able to move on a begin comparing different clusters to see if there is an overall similarity in nucleotide sequence between tape measure proteins.

*We are predicting some similarity due to the fact that they are all tape measure proteins; therefore, they share similar morphology. Having a similar morphology means that there is a similar average tail length.

 

Ashton Peckinpah

Tuesday, March 21, 2017

Objective: To begin conducting our independent research projects. We will continue to make any changes if necessary based on our findings. We will plan to further explore bioinformatics tools online and use them if necessary in supporting our research.

Methods: 

1. Identify the pham of the tape measure gene in Courtney3.
2. Collect the protein and nucleotide sequence of the Courtney3 tape measure protein.
3. Identify and examine other published genes within that pham.
4. Collect protein and nucleotide sequences of the tape measure proteins for other published genes within the pham.
5. Compare these sequences by aligning them on Gepard.

Tools Used: DNAMaster, Phamerator, and NCBI Blast

In conclusion: Using Phamerator, we found that the Courtney3 tape measure protein was in pham 6177. There were several other published genomes within this pham: these included, Maggie, Decurro, Chestnut, Moloch, and Multtie.

After researching all of these genes, we found that all genes within a particular pham have a 100 protein and nucleotide sequence alignment. Since examining this, we determined that all phams share the same sequences for a gene with the same function (in this case, the tape measure protein).

*We determined that all phams share the same sequences for a gene with the same function (in this case, the tape measure protein). After recognizing this similarity, we decided to begin comparing different clusters of sequences. By comparing different clusters, the genomes will have different tape measure sequences, so they will not match identically as do phams.

After doing this basic research, we better understood the categorizing of phages. From this point moving forward, we will have to redesign our research project. Instead of comparing sequences between phams, we will examine multiple tape measure sequences in different phams.

 

Ashton Peckinpah

Tuesday, March 14, 2017

Objective: To understand different softwares and additional bioinformatics research tools that can be used during our independent research projects. Moreover, we branched off into groups and began the brainstorming process for our final research project.

Methods:

1. Explore phagesdb.org glossary and links in order to understand different terms and tools that can be used during our project.
2. Explore outside genome research tools.
3. Decide on a proposed research question.
4. Decide which tools will be used to research this topic.

In Conclusion: We plan to explore sequence similarities between various tape measure proteins. Our goal is to see if there is a similar sequence within each gene that is present in all phages. After reviewing phagesdb.org, we learned that the tape measure gene is the longest gene within the genome, and it is directly correlated with the length of the proteins tail.

 

Ashton Peckinpah

Tuesday, February 27, 2017

Objective: To annotate the entire Timinator genome by having each student annotate five genes. This allowed for each student to feel more comfortable annotating genes as we approach the beginning of our research projects. Today, I was given the challenge to annotate assigned genes for Timinator. I was assigned:13, 35, 57, 80, and 68.

(Tools Used): DNA Mastering, GeneMark, Phagesdb, Starterator, Phamerator, Google Docs, and HHPred

Methods: First, I had to run an auto-annotation of the Timinator draft in DNA master. For each gene assigned, I had to complete each of the following portions of the annotation:

• SSC:/ Gap or overlap: start/ stop and gap/ overlap distance.
• CP: covered by all.
• SCS: Does it agree with Glimmer/ GeneMark.
• NCBI Blast: Best blast hit, E-Value, Conserved Domain hit, and CDD E-value.
• SD: SD score, ranking, and Z-value.
• PhagesDB Blast: Best blast hit, E-Value.
• HHPred: Best blast hit, E-value.
• LO: longest open reading frame.
• ST: does it agree with Starterator.
• F:/ FS: function and support for this function.

After completing the above portions of the annotation I  inserted the annotation components into the Timinator Google document. Every time I had to update the status on the home page and confirm that the gene annotation was/is complete.

Results: 

Gene 13-

Start: 9980bp Stop: 10591bp FWD GAP: 0bp Gap SD Final Value: SD Score: -3.467 (Best score) Z-Value: 2.701 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein Arthrobacter phage BarretLemon_13 E-Value: 0.0 CDD: No good hit PhagesDB BLAST: DarthPhader_8, function unknown, 307 E-Value: 2e-10 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, PhagesDB Notes:

(Phagesdb)

(RBS table results)

 

Gene 35-

Start: 29826bp Stop: 30671bp FWD GAP: 2bp Gap SD Final Value: SD Score: -2.275 (Best score) Z-Value: 3.294 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: RecT-like recombinase [Arthrobacter phage BarretLemon] E-Value: 0.0 CDD: RecT Superfamily E-Value: 9.89e-98 PhagesDB BLAST: StevieBAY_Draft_35, function unknown, 281 E-Value: e-161 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: Phages DB Notes: NCBI said the function of this gene was a RecT DNA recombinase, but this function option was an option in PhageNotes.

(C0nserved Domain)

 

Gene 57-

Start: 40263bp Stop: 40526bp FWD GAP: 4bp Overlap SD Final Value: SD Score: -6.513 (Best score) Z-Value: 1.478 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: HTH DNA binding protein [Arthrobacter phage BarretLemon] E-Value: 8e-42 CDD: No good hit PhagesDB BLAST: StevieBAY_Draft_57, function unknown, 87 E-Value: 3e-42 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: HTH DNA binding protein, MerR-like FS: NCBI Notes:

(RBS results)

 

Gene 68-

Start: 43977bp Stop: 44168bp FWD GAP: 4bp Overlap SD Final Value: SD Score: -3.697 (Best score) Z-Value: 2.625 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein BARRETLEMON_67 [Arthrobacter phage BarretLemon] E-Value: 8e-38 CDD: No good hit PhagesDB BLAST: LeeroyJ_Draft_68, function unknown, 76 E-Value: 2e-36 HHPred: No good hit LO: No Better SD score ST: F: NKF FS: NCBI, PhagesDB Notes:

(RBS Result)

 

Gene 80-

Start: 50855bp Stop: 51088bp BKWD GAP: bp Gap SD Final Value: SD Score: -6.658 (Best score) Z-Value: CP: The gene is covered SCS: Disagrees with Glimmer, Agrees with GeneMark Genemark gave me a better SD score NCBI BLAST: hypothetical protein BARRETLEMON_79 [Arthrobacter phage BarretLemon] E-Value: 1e-47 CDD: No good hit PhagesDB BLAST: StevieBAY_Draft_79, function unknown, 77 E-Value: 1e-39 HHPred: No good hit LO: Yes ST: F: NKF FS: NCBI Blast, PhagesDB, HHpred Notes:

This gene was deleted (per professor) since it had a 100% overlap with gene 79.

Additionally, the gene had no known function.

In conclusion: We were able to put all of our semester work to the test. As a class, we successfully annotated the entire genome of Timinator. Now, it can be sent off to Pittsburg to be approved.  This annotation will be useful in the future when researching gene functions, promoters, and other parts of the genome.

Ashton Peckinpah

1/10/2017

Objective: to familiarize myself with the background into phage biology investigation, and the purpose of the laboratory

Methods: We were placed into pairs so that we could further investigate important applications of phage therapy. The assignment was to put together a quick powerpoint presentation that summarized the abstract of the project and also to include a picture to analyze.

Results: My partner and I were assigned to the bacteriophages that were found in specific genomes. After looking through the abstract, we found the article to mainly consider different clusters within a genome and determining if there are known or identifies similarities or differences.

In conclusion: This lab was successful since it introduced us to the role we will be playing throughout the course of the year, and the potential our work could have on future research.

Lab 2: Genome Annotation

Ashton Peckinpah

1/17/2017

Objective: Today we familiarize ourselves with the tools we will be using for the remainder of this semester. One being DNAMaster used to auto-annotate the genes and read the results of the annotated genomes.

Methods: After watching a few DNA Mastering guided videos, we walked through the process with the phage Amigo.

1. We were first taught how to download DNA Mastering onto our own individual laptops.
2. We used the DNA Mastering annotation guide to walk us through the process of annotating a genome.
3. We then began setting up our preferences on DNAMaster:

-Click on the File menu within DNAMaster and select Preferences

-Click on Local Settings tab and Colors sub-tab. We changed the colors to help us decipher and identify differing RNA’s.

-Click on Codons sub-tab and make sure “Use TTG start codons” is checked.

-Template code SSC: CP: SD: SCS: Gap: Blast: LO: ST: F: FS: ST

-May change SD scoring matrix to Kibler 7 and the spacing weight matrix to Kerlin medium

-Click the Apply button

4. Used phagesdb.com to download the FASTA file of the phage amigo.
5. Then we opened the new FASTA file amigo and auto annotated it.

Auto-Annotation:

1. Find Amigo file and click Open.
2. Go to Export button and click “create Sequence from this entry only”
3. From the “Extracted from Amigo.fasta” window, go to the Genome menu and select Annotation, Auto-Annotate.
4. Click Annotate.
5. Once your genome is annotated, click on the Features tab at the top of the genome’s window.
6. Right click the “Name” row and expand the row.
7. Click ORF’s button.
8. Click on an ORF.
9. Click on the button labeled RBS.

In conclusion: After completing the lab, we learned how to properly use DNA Master. We begin to understand the information we are looking at especially after having this good foundation for to start with.

 

 

 

 

 

 

 

Lab 3: Link BLAST

Ashton Peckinpah

Tuesday, January 24, 2017

Objective: Understand how to complete a BLAST, which can be considered a critical element of a gene annotation. The BLAST tool finds regions of similarity between the sequences and generates a report to show the comparison.

Methods:

1. Download the Link fasta file from phagesbd.org.
2. Open DNA master.
3. Open file in DNA master.

4. Copy and Paste the product of the amino acids of the gene you want into NCBI protein blast.

1. Add the following annotation notes in preferences: SSC: CP: SD: SCS: Gap: Blast: LO: ST: F: FS:
2. BLAST

-Copy and paste the code in the product window.

-Go to the protein blast option on NCBI and PhagesDB.

-Paste the code there.

-Run the gene.

-View results.

Results: there is a list that follows the colorful graph. The list shows the most and least similar genes

When learning what the annotation template codes stand for, refer back to the DNA Mastering Annotation Guide.

-SSC: start/stop codons

-CP: codeine potential

-SD: Shine Dalgarno Score (also includes z value)

-SCS: whether or not the start codon choice agrees with glimmer and gene mark

-Gap: the gap or overlap with the gene before it

-Blast: Blast on NCBI, HHPred, phages DB

-LO: longest ORF

-ST: starterator

-F: Function

-FS: Function Source

In conclusion:                            

We now understand how to use GeneMark to indicate if a gene is entirely covered as well as the coding potential following the gene. For me, I better understood how to annotate a genome after blasting it. Knowing that when an individual blasts, they are simply comparing one genome to another.

As a class, we annotated genes 7 and 8. The GeneMark potential covered the entire genome, the starterator agreed with the start site, and the genes showed the best SD score as well as the longest ORF.

I feel prepared to annotate genes in the future after what knowledge was gained through this lab.

 

 

 

 

 

 

 

Lab 4: Phamerator

Ashton Peckinpah

Tuesday, January 31, 2017

Objective: Understand how to use Phamerator correctly as well as incorporate the useful technology into a genome annotation.

Methods:

Phamerator:

1. Open the Virtual Box application.
2. Click on the Phamerator option.
3. Click on the phages tab to the left.
4. Search link and then click map (6 genomes when comparing)
5. Note the Pham number, members, and cluster.
6. Go back to the phages tab and select several other AN Arthrobactors.
7. Once you have clicked on phages you need, Click map.

Annotations:

-Annotate assigned gene.

-BLAST (NCBI and PhagesDB)

-Observe conserved domains of NCBI

-Observe Phamerator results.

-Function of the genome?

*Understanding how to read a phamerator report is a very important part of this lab

In conclusion:

Phamerator compares the genes using multiple alignments. Overall, Phamerator helps to clarify our genes function by comparing it with other phams (usually similar).

 

 

Lab 5: Starterator

Ashton Peckinpah

Tuesday, February 7, 2017

Objective: Learn how to operate Starterator, this piece of technology may help clarify our start codon on our genes. It is helpful in an entire genome annotation.

Methods:

Starterator:

1. Open the Virtual Box.
2. Hit start and open Phamerator.
3. Click on the phages tab and search for the phage of your choice.
4. Click map.
5. Note the Pham number, members, and cluster.
6. Compare the recommended start site (indicated with blue) to that of Glimmer and GeneMark.
7. Indicate if you agree with start recommended by Starterator.
8. There will be a graph followed by a text summary

How to read Starterator:

1. each track represents an ORF
2. Each colored line represents a possible start codon
3. the white lines represent gaps.
4. The report lists the tracks and the names of the genes and suggested starts Results: for gene 7 Lore_draft

 

In conclusion:

Starterator is one last tool used to accurately identify correct start codons. This tool is really helpful and I know it will be helpful next week with my lab partner and I.

 

 

 

 

 

 

Lab 6: Annotating Lore Genes 23 & 25

Ashton Peckinpah

Tuesday, February 14^th, 2017

Objectives: Beginning to work on the gene Lore today after weeks of preparation. I plan on completing annotations for gene 23 as well as 25 today.

Tools used and/or Methods: Glimmer, GeneMark, HHPred, NCBI, PhagesDB,  Phamerator, Starterator

Results for Gene 23: Start: 14113bp, Stop: 14781 bp FWD GAP: 4bp Overlap

GeneMark confirmed that all potential, typical as well as atypical, is covered.

NCBI Blast identified an HTH superfam. Confirming it supports HTH DNA binding function, q:1 s:1

 

Phages DB blast: no known function for the gene

HHPred: supporter of HTH DNA binding function

 

Final Annotation Result:

Start: 14113bp Stop: 14781bp FWD GAP: 4bp Overlap SD Final Value: SD Score: -3.246 (Best score) Z-Value: 2.847 CP: The gene is covered SCS: Agrees with Glimmer, Disagrees with GeneMark Genemark called a reverse gene, but there was more potential in the forward gene that was not called NCBI BLAST: Helix-turn-helix DNA binding domain protein [Arthrobacter phage Decurro] q:1 s:1 E-Value: 2e-156 CDD: pfam13730, Helix-turn-helix domain E-Value: 1.09e-06 PhagesDB BLAST: StewieGriff_Draft_23, function unknown, q:1 s:1 E-Value: 1e-134 HHPred: Transcriptional Regulator E-Value: 5.9E-08 LO: Yes ST: Agrees with Starterator F: helix-turn-helix DNA binding domain FS: NCBI, HHPred, Phamerator Notes:

 

 

 

Results for Gene 25:  Start: 14980bp Stop: 15276bp FWD GAP: 4bp Overlap

GeneMark confirmed that all potential, typical as well as atypical, is covered.

NCBI Blast: no known function for gene

Phages DB Blast: no known function

HHPred: no known function. Too high of an e value for the top hit.

 

 

Results for Gene 25:

 

Start: 14980bp Stop: 15276bp FWD GAP: 4bp Overlap SD Final Value: SD Score: -3.554 (Best score) Z-Value: 2.798 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein STRATUS_25 [Arthrobacter phage Stratus], q:1, s:1 E-Value: 4e-46 CDD: No good hit PhagesDB BLAST: StewieGriff_Draft_25, function unknown, q:1 s:1 E-Value: 2e-51 HHPred: No good hit LO: No All coding potential was covered with start position 14980 and LO had lower final score ST: Agrees with Starterator F: NKF FS: NCBI, Phages DB, HHPred Notes:

 

 

 

Lab 7: LORE Presentations

Ashton Peckinpah

Tuesday, February 21, 2017

Objective: Today we presented the genes we annotated so others may see and critic them as well. We also see what an entire genome looks like. As a class, we deciphered if a start codon change was necessary or if declared functions were correct.

Methods:

1. Groups presented PowerPoint presentations to class.
2. For others in the class not presenting, fill out the function.

-Was it auto-annotated?

-Were there any changes made to Lore?

3. You fill out the Questions That Matter worksheet with the above information.
4. We listened intently to others presentations. It helped me understand the annotation process listening to others experiences doing it.

In Conclusion:

This lab taught us how to successfully annotate an entire genome. Afterwards we were happy with our accomplishment of completing our first phage. It is exciting to think about these getting double-checked and then getting sent off to be published.

We begin annotating our next phage next week.