Goals: Form groups for making URSA posters and begin working on the poster.

Methods: Because our groups needed at least one representative from each of the genome teams (Shrooms, Nubia, and Caterpillar), I formed a group with Chrissy, Andrea and Stu. We began the planning of our poster on 3/15 and finished our poster on 3/20.

Our Poster:

Poster-vp0d5z

Next steps include presenting our poster to the class and picking two to print for URSA Scholars Day!

Goals: Finish the cover sheet (annotation summary) and assist group in finishing the review process.

Finished Annotation Summary:

Arthrophage Shrooms Annotation Summary

Michael Munson, Stu Mair, Natalie Widdows, Niharika Koka, Emily Johnson,

Andrea Springman, Niru Ancha, Daniel Zeter

Baylor University

Totals:

Gene Count: 98

Insertions: 1

Deletions: 3

Extensions: 11

Reductions: 1

No Known Function (NKF): 67

Identified Functions: 31

Debatable Calls:

Gene 28 – The gene was extended to 20600bp to cover all the coding potential. The extension agreed with Starterator, NCBI BLAST, and PhagesDB BLAST. Extending the gene also improves the Z value from 2.771 to 2.052. This call does, however, disagree with both Glimmer and GeneMark and sacrifices a better SD score. Glimmer and GeneMark called the start at 20624bp, and the SD score changed from -3.682 to -5.306.

Gene 34 – The gene was extended to 22385bp to improve the SD score from -5.632 to -4.759 and the Z value from 1.157 to 2.13. Extending the gene did, however, conflict with GeneMark, Glimmer, and Starterator which called the start at 22397bp. While both PhagesDB BLAST and NCBI BLAST had results, neither extending the gene nor leaving the gene aligned the start codon with the compared genomes. NCBI: Q9, S3; PhagesDB: Q10, S4.

Gene 48 – The gene was extended to 27837bp to cover all the coding potential. This call does, however, conflict with both Glimmer and GeneMark which called the gene at 28040bp. There were no hits for this gene on NCBI BLAST or PhagesDB so making the call was difficult.

Conclusions: After finishing the annotation summary, I helped the rest of the group finish the peer review because our group was struggling to get through all of the genes. But we all finished, and we were able to submit all of our work and final DNA Master files to Dr. Adair for her review. All that’s left is for the genome to be submitted. Yay!

Goals: Delegate tasks within our group to begin the review process for the Shrooms genome that we have annotated.

Our tasks are:

• Proof each of the annotations to make sure the calls were good and are free from error
• Write a cover sheet explaining difficult calls and giving an overview of the genome
• Make a final DNA Master file with annotations pasted in to the notes section
• Make a DNA Master file of all of the assigned functions

We discussed these as a group and delegated the tasks. It was decided that I would work on the cover sheet. During lab I began working on this, although I was not able to finish. I began to compile all the information about the number of changed starts, insertions/deletions, difficult calls, etc.

Future goals include finishing the annotation cover sheet.

Goal – Annotate genes 44, 46, 48, and 50

Tools – Phamerator, Starterator, DNA Master, GeneMark, Glimmer, NCBI BLAST, PhagesDB BLAST, HHpred

Results:

Gene 44 –

Start: 26673bp Stop: 26855bp FWD GAP: 4bp Overlap SD Score: -6.571 (2nd best score) The best score would have caused too big of a base pair overlap with the previous gene Z-Value: 1.08 CP: The gene is covered SCS: Agrees with Glimmer, Disagrees with GeneMark Genemark did not call the gene. NCBI BLAST: hypothetical protein LAROYE_46 [Arthrobacter phage Laroye]; Q30, S98 E-Value: 4e-10 CDD: No good hit PhagesDB BLAST: Laroye_46, function unknown; Q30, S98 E-Value: 1e-12 HHPred: No good hit LO: No Longest ORF would cause an overlap of 87bp ST: Agrees with Starterator F: NKF FS: NCBI, Phamerator, HHPred Notes:

Coding Potential Gene 44

NCBI BLAST 44

Gene 46 –

Start: 27430bp Stop: 27849bp FWD GAP: 299bp Gap SD Score: -4.18 (Best score) Z-Value: 2.576 CP: The gene is covered SCS: Disagrees with Glimmer, Agrees with GeneMark NCBI BLAST: No good hit CDD: No good hit PhagesDB BLAST: Waltz_Draft_46, function unknown; Q1S1 E-Value: 3e-69 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, Phamerator, HHPred Notes:

PhagesDB BLAST Gene 46

Gene 48 – 

Start: 27837bp Stop: 28040bp FWD GAP: 13bp Overlap SD Score: -4.537 (2nd best score) The best score does not cover all the coding potential. Z-Value: 2.476 CP: The gene is covered SCS: Disagrees with Glimmer, Disagrees with GeneMark These calls left uncovered coding potential. NCBI BLAST: hypothetical protein SALGADO_37 [Arthrobacter phage Salgado] E-Value: 6e-46 CDD: No good hit PhagesDB BLAST: Edmundo_Draft_50, function unknown, Q4, S1 E-Value: 6e-26 HHPred: No good hit LO: No Longest ORF would cause an overlap of 193bp ST: Starterator was basing this call only on drafts and therefore was not very reliable. The gene was extended against Starterator to cover the coding potential F: NKF FS: NCBI, Phamerator, HHPred Notes: The start codon called by both GeneMark and Glimmer left uncovered coding potential. I chose to extend the gene to cover the coding potential.

PhagesDB BLAST Gene 48

Gene 50 – 

Start: 28231bp Stop: 28398bp FWD GAP: 4bp Overlap SD Score: -5.401 (2nd best score) The best score has an ORF length of 63 bp. Z-Value: 1.624 CP: The gene is not covered There is no start codon by which to extend the gene to cover the coding potential. SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: No good hit CDD: No good hit PhagesDB BLAST: Waltz_Draft_48, function unknown, Q1 S1 E-Value: 7e-19 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, Phamerator, HHPred Notes:

PhagesDB BLAST Gene 50 –

Coding Potential Gene 50 –

Conclusions: Today Nihi and I finished annotating our portion of Shroom’s genome. The rest of our team has just about finished their portion too and so the next task is to review the annotations, write a cover sheet, and form a final DNA Master file for submission.

Goal: Make powerpoint presentation

Results: We completed our powerpoint and presented to Ashley, who critiqued us. We made the changes she recommended. Next all we need to do is practice and present this Friday!

Independent project finished!

Goal: Write Abstract for Presentation

Abstract – 

Analysis of Alternative Clustering Methods for Arthrobacteriophages

The study of bacteriophages includes an investigation of their DNA. An important aspect of DNA analysis is clustering, the means by which discovered bacteriophages are sorted according to genome similarity. The current method for clustering is an analysis and comparison of a bacteriophage’s full genome. However, this presents several limitations for clustering, as it requires the completion of full-genome sequencing and the production of a high-titer DNA lysate. In the interest of easing the clustering process, additional clustering methods were analyzed including sorting by G/C content and clustering by Tape Measure Proteins (TMP). Through comparison to existing clusters, it was determined that the G/C content of phage clusters group too closely to be useful for clustering. However, through the use of a Gepard dotplot, it was determined that there is enough similarity among TMPs within clusters and enough diversity between clusters to facilitate that clustering method. From this, we used cluster TMP alignments generated by Mega7 to find conserved regions within each genome. From these, small PCR primers were created that were specific to each cluster’s TMP with minimal off-target similarity. These will allow future researchers to determine the clusters of bacteriophages with a minimal sample of DNA earlier in the process without going through whole genome sequencing. In conclusion, TMP analysis is a highly effective alternative clustering method with the added benefit of PCR primers, which offer the benefits of less lab work and the ability to sort phages with a low titer DNA lysate.

Conclusions: With the abstract finished, all we have left to do is to make the presentation!

Goal: Begin brainstorming about how to use PCR primers to cluster.

Discussion: In the article referenced for the use of TMPs to cluster, the authors designed PCR primers for the TMP proteins. We discussed that we can do this with our phages, designing primers unique and specific for each of the 13 clusters. This way, we can cluster without whole genome sequencing and without having a high titer lysate. Additionally, this process could be performed in the wet lab to see the cluster of all the phages that don’t make it to sequencing.

What we did: 

First, we collaborated with another group with whom we had similar topics. We decided to diversify – we are going to focus more on TMPs and designing PCR primers.

Next, we continued researching how we can design primers. We learned the primers need to be approximately 20 bp. We need both a forward primer and a reverse primer. The primers must be distinct enough that there is no more than 80% similarity in the other clusters.

Goals for Next Lab: 

Begin designing the PCR primers for all 13 clusters.

Goal: Look at the G/C content in each of the 39 genomes of the 13 clusters and see if the G/C content is conserved and if it can be used to cluster.

Methods:

Compile GC content info on all 39 phages

Create graph in Excel to visualize the clusters

Results:

Conclusions: 

G/C content, while highly conserved within each cluster, is very similar across clusters and therefore cannot be used to cluster. Next time we will begin to look at the possibility of designing PCR primers for the TMP proteins.