Link to this post | posted 06 May, 2018 20:55
cdshaffer	The updated phamerator database has now been fully run though starterator and the results have been posted. You will now find your report available: http://phages.wustl.edu/starterator/Pham45669Report.pdf Edited 07 May, 2018 03:12

Link to this post | posted 26 Apr, 2018 20:33

cdshaffer

Just a note in case someone comes to this thread later, in cases where the pack/unpack protocol doesn't work. Before you start over from scratch to redo all your annotations in a new DNA Master file you can try to at least transfer the notes and annotations using the technique Welkin describes in this post: https://seaphages.org/forums/topic/4547/?page=1#post-6264 That technique has saved me many times.

Link to this post | posted 26 Apr, 2018 16:00

cdshaffer

An HHpred search shows weak evidence for a terminase with matches of 95% (e .11) to COG3747; Phage terminase, small subunit, and to PF05119 Terminase_4 Phage terminase, small subunit 94% (e .24). The issue here is you won't find those if you use the default PDB database, you have to add the other databases before you do the search. See this page for the list of databases: https://seaphagesbioinformatics.helpdocsonline.com/article-19 I would say the evidence is pretty weak on its own but if you look at this page: https://seaphagesbioinformatics.helpdocsonline.com/article-91 you will see that at least one but no more than two terminase proteins must be present somewhere in your phage's genome. So annotation of this gene probably depends on if you have better evidence that some other gene or genes have stronger support for being a terminase, in which case one would probably argue that this protein is, as you suggest, a distant relative of a terminase but is not THE terminase. On the other hand if no other protein in the genome has even a hint of being a terminase then I think that combining the expectation that there must be a terminase somewhere with the hhpred hits is strong enough to justify the terminase annotation.

Link to this post | posted 20 Apr, 2018 16:59
cdshaffer	Working link: http://www.techrepublic.com/blog/windows-and-office/selectively-disable-uac-for-your-trusted-vista-applications/

Link to this post | posted 19 Apr, 2018 05:32

cdshaffer

I must say I really like this gene cluster. I use it as a great didactic opportunity to have students think carefully about biochemistry and the nature of enzymes. I remind them that all an enzyme can do is speed up the reaction rate, it cannot control "the direction" of the reaction. The direction depends on which substrates are in excess. This is especially an issue when enzymes are purified and studied in vitro where substrate concentrations can be orders of magnitude different than intracellular levels. This leads to confusion when trying to functionally annotate and this cluster is a great example. The top hit from hhpred is called an RNA Ligase on the short text but when you dig into the enzyme it all makes sense. The pdb entry for this RNA Ligase is 1IUH. Here is the link: https://www.rcsb.org/structure/1iUH If you go to the page and click on the "annotations" tab you will see down in the "molecular function" section that this one enzyme lists both an RNA ligase and a 2' 3' Cyclic Nucleotide 3' Phosphodiesterase activity are listed. This is because experimenters can detect both activities in vitro. Some labs put in the cyclic nucleotide and see breakage of the phosphate to carbon bond (phosphodiesterase). A different set of substrates and you can drive the reaction the other way and create a phosphate to carbon bond (ligase). As for annotation, of the available approved terms, I feel like RNA ligase is the best available term since phosphodiesterase is not available. Phosphoesterase just feels too generic since there are many phosphoesterases that are not phosphodiesterases.

Link to this post | posted 23 Feb, 2018 20:24

cdshaffer

It is not unusual to get slightly different results from gene predictors when the same sequence is analyzed on different computers. Software version, default parameter settings, different training sets and other factors can effect the outcome. This is really just par for the course in bioinformatics. Without good evidence to the contrary I always treat each source (PECAAN, DNAMaster, Phamerator) as evidence to be used in the final annotation. Those genes that appear in one auto-annotation and not the other simply have less evidence to support them being in the final annotation. From the Guiding Principles of Bacteriophage Genome Annotation rule 2 we know that genes rarely overlap by more than 30 bp, so clearly some of those genes in that cluster as displayed in the phamerator map should not end up in the final annotation. So the genes in that region that show up in both have slightly more evidence supporting their existence compared to the genes that only show up in one. However, you will want to use all sources of evidence before you decide which genes should end up in your final annotation. See Deciding whether an auto-annotated gene is a gene.

Link to this post | posted 13 Feb, 2018 18:24

cdshaffer

Unfortunately the last build of the SEA VM was done before I was able to release the version of Starterator with all the bug fixes. So the SEA VM version will work for many but not all phage. I have run a full phage report for Hegedechwinu on the updated version of starterator and you can download it from here: https://wustl.box.com/s/16md6n7clzkwz13xdvfpabxu8c28gjp5 Feel free to download and use. Be aware that what might work better for you is to use the pre-computed starterator reports. The nice thing about these reports is they are automatically updated with each new release of the phamerator database. These pre-computed pages are part of the effort to get away from the VM and get as much as possible available via web browser. These reports are available as links off of the phagesdb pages for the phams and the individual gene pages. Examples for pham 431: http://phagesdb.org/phams/431/ (Pham page, click on the blue “Get Starterator Report” button at the top of the page.) Example for gene 9 of phage Pollywog: http://phagesdb.org/genes/POLLYWOG_DRAFT_9/ (Gene page, click on the link “Pham 431 Report”, fourth row of the table.) Alternative methods for obtaining the reports are discussed on this forum page well. Edited 14 Feb, 2018 01:31

Link to this post | posted 13 Feb, 2018 18:00

cdshaffer

Not every gene that codes for a protein will show strong GeneMarkS potential. For a discussion of this see the forum page on Typical vs atypical GeneMarkS coding potential: https://seaphages.org/forums/topic/75/ But the bottom line is that Coding potential is based on looking at very long orfs in the genome itself to determine the sequence characteristics of a "typical" gene, it then uses that definition to score every region of the genome to indicate how closely it matches the "typical" result. Not all genes are expected to be "typical" so lack of coding potential is not good evidence for lack of a gene. See the discussion posted above for more details. As for Starterator, I have reposted your question to the Starterator section of the forum: https://seaphages.org/forums/topic/4500/ Edited 13 Feb, 2018 18:16

Link to this post | posted 13 Feb, 2018 17:54

cdshaffer

I am cross-posting this request for starterator help from grosasacosta below: I am having issues with Starterator. I've been trying to run it for Hegedechwinu but it keeps crashing when it reaches Pham 92 (near the end of the analysis). Any suggestion on how to solve this issue? It is related to that specific Pham. From what I saw in other similar posts, the solution may not be simple at all. Please let me know if you have any suggestions on how to solve this issue, I will be waiting. Edited 13 Feb, 2018 18:04

Link to this post | posted 08 Feb, 2018 23:30

cdshaffer

This is a general announcement that the starterator version used to create the pre-computed pham reports is being changed as of Feb 8th. This is necessary due to the ongoing work behind the scenes to improve the phamerator database. These changes to the database have necessitated updates to the methods starterator uses to determine if a phage should be considered a "draft" annotation. Most of the changes you will notice are cosmetic in nature, and feedback on these are always appreciated. More importantly, it is possible that the changes to the underlying code may have inadvertently introduced errors in determining if a phage is indeed "draft" quality. Please be on the lookout for errors of this type as you use the pham reports. If you find any examples which you believe may be in error please post these in this thread. Edited 09 Feb, 2018 17:28