Link to this post | posted 25 Jan, 2018 18:07

cdshaffer

It is technically possible to pre-computed whole phage reports for all phage but it is currently unfeasible. Whole phage reports take on average about 5 minutes on a decently powered computer and there are currently over 2300 phage. That works out to about 1 week to calculate all reports. Given that there is a new database every week means I would need 1 computer running full time and totally dedicated to the task. As for Starterator, there is the current "Stable" version that I used to create the report posted above. There is also an "Experimental" version that has a bunch of changes to the output. For comparison here is the output from the "experimental" version: Pollywog_DraftReport_experimental.pdf The experimental version has some improvements that I really like (e.g. sorting of the phage track to the top) that will certainly be in the next "Stable" release, but it also has some parts that still need work (e.g. like the new summary table) and will definitely be changing as soon as the semester calms down and I can get back to coding. Let me know which version you are interested in, and we can go from there.

Link to this post | posted 24 Jan, 2018 20:52

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 pollywog on the updated version of starterator and you can download it from here: Starterator report on Pollywog. Feel free to download and use but it may not be what would work best for you depending on your didactic goals. If you want include the use of VM's and Starterator in your class then it is probably best to update the VM with the latest version of starterator. It is fairly straight forward, just post a follow-up query on how to update starterator if you are interested. Another alternative is to use the pre-computed starterator reports. This is part of the effort to get away from the VM and get everything available via web browser so it is platform independant. These reports are available as links off of the phagesdb pages for the phams and the individual gene pages. Examples: http://phagesdb.org/phams/431/ (Pham page, click on the blue “Get Starterator Report” button at the top of the page.) http://phagesdb.org/genes/POLLYWOG_DRAFT_9/ (Gene page, click on the link “Pham 431 Report” link in the fourth row of the table.) The good part about these pre-computed reports is that they are updated every time new phage genomes are added to the phamerator database throughout the spring. The whole phage report above may get out of date if more F1 phages are sequenced and added to the database.

Link to this post | posted 10 Jan, 2018 22:26

cdshaffer

DNA Master prefereces must now be updated to use the PBI servers specific for SEA-PHAGES courses. See the help document, it is in the faculty only -> faculty info page, in the Bioinformatic Component section. The link is called "DNA Master Gene Prediction Settings", it describes how to change a few preference settings in DNA Master to get auto annotation working. If you followed those instructions and it is still failing, check your internet connection.

Link to this post | posted 10 Jan, 2018 19:58

cdshaffer

Yes, I think you are correct. I have found DNA Master to be a bit finicky. I had one student last semester with a mac and we were never able to get it to run successfully even though it was running just fine on all the other macs in the class. Like you, we tried deleting everything and reinstall several times with no luck, it just never worked on that one mac. I tried to replicate your issue on my mac and was able to import and auto-annotate a C phage, so like my one student you are probably doing everything correctly and it is just something with the macs you are using. The only suggestions I have, give that the issue only appears with larger genomes, would be to try macs with more memory, it's a long shot but might work.

Link to this post | posted 09 Jan, 2018 18:06
cdshaffer	If you are getting the access violation when trying to auto-annotate check out the DNA Master troubleshooting guide here: http://phagesdb.org/DNAMaster/faq/ If it is happening somewhere else, could you give more details on exactly which steps you are taking and what you did just before the got the error.

Link to this post | posted 15 Dec, 2017 20:40

cdshaffer

Analysis of protein OzzyJ_38 draft annotation. When we run an HHPRED analysis at the original site, the top hit is to a crystal structure with 99.18% probability and an e-score of 2.6x10-12 to pdb crystal structure 2WCW which is described as a Holiday junction resolvase from Archaeoglobus fulgidus. At this point looking into the issue of it was a "RusA" or "RuvC" type of resolvase we looked at the 4th best HHPRED hit (which actually had an associated published paper) with a probability of 98.81% and e of 4.9x10-10 to the pdb crystal structure for 1GEF. This is also described as a Holiday junction resolvase but was from the hyperthermophilic archaeon Pyrococcus furiosus. The paper describing these results (Structure. 2001 Mar 7;9(3):197-204) describes this structure as "The folding of the Hjc subunit is clearly different from any other Holliday junction resolvases thus far known." Instead the overall structure is described to be similar to type II restriction endonucleases. For now, we have submitted this protein with the simple annotation of "holliday junction resolvase" since it is neither a RusA or RuvC type. However, if adding a type was preferred, adding a descriptor like "Archaeal" or some indication of its similarity to type II RE enzymes might be appropriate. Follow on thoughts appreciated.

Link to this post | posted 07 Dec, 2017 16:32

cdshaffer

Chelsea, As a back up plan students can enter their data directly at phagesdb.org. Ideally students should upload EM, digest, and plaque pictures (if they have them). To prepare images for upload see this: http://phagesdb.org/media/workflow/protocols/pdfs/MakingThumbnails.pdf Also, before going to uploading everything, go get the GPS coordinates using Google maps. Just go to google maps and zoom in to the point where you can accurately click on the collection site, after clicking exactly on the spot in the map a box will show up with the decimal GPC coordinates, just copy them for the last step. Go to the add a phage page on phagesdb.org which is here: http://phagesdb.org/addphage/ Or if the phage is already been added to phagesdb.org they can go here to make updates: http://phagesdb.org/modphage/ Students should fill out as much as they can but some fields will certainly be left empty as they require the genome sequence. I especially encourage my students to fill out the Naming notes section; mostly because I am always curious the how and why of a particular phage name.

Link to this post | posted 29 Nov, 2017 16:45

cdshaffer

I think this issue of the phamerator database synchronization is the most pernicious problem out there. I have been trying to think of ways to improve the system but it is tricky given the various distributed programs that use the database. I keep some older versions of the database just for historical records. It looks like the last version of the database with pham 18287 was version 117. You can see the starterator report here (note the 117 in the URL: http://phages.wustl.edu/117/Pham18287Report.pdf That pham is not found in my next stored version (120), with that version the gene Kareem_11 has moved to pham 29797: http://phages.wustl.edu/120/Pham18287Report.pdf When using the SEA VM the only way to update to the most recent version of the phage database is to run phamerator. Once you start phamerator it will check in to see if there is an update, and if so, it will attempt to download and install. Once you get a successful database update then the SEA VM Starterator will match the current online versions.

Link to this post | posted 14 Nov, 2017 00:12

cdshaffer

This is a proposed funtion based on Pham 28735 (example member Mildred21 gp255) The HHPred has a 100.0%/e=9E-39 93% length alignment to an NMR structure to e coli YibA in PDB and to COG3236 see screen capture of hhpred alignment here. The phyre2 3d prediction is a 100% confidence with 91% coverage to the same pdb structure. This link should work for about a week. here is the screen capture which should work for the foreseeable future. YibA is a founding member of COG3263, the function for COG3263 is based mostly on this paper: A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis. Biochem. J. 2015 Feb 15; 466(1):137-145 This paper clearly shows that this e coli protein (and a couple of others like it) can enzymatically function to cleave N-glycosidic bonds in vitro as part of a large complex that carries out the first few steps of riboflavin biosynthesis. So there is really good evidence for enzymes of this type to be able to hydrolyse N-glycosidic bonds. I am certainly not proposing that this enzyme in phage is involved in riboflavin biosynthesis, but the wet bench is pretty good that the e coli YibA does have a glycosylase activity and there are many different glycosylases. The issue as to naming is one of specificity, should we use the more specific N-glycosylase or the more generic glycosylase? There are also S, O and C- type glycosylases. I feel that without biochemical evidence or a published analysis of the amino acids present at the active site and how they interact with a particular glycosidic bonds (i.e. how does the active site change with N, S, C and O type glycosidic bonds) that specifically adding the term N-glycoslyase may be too specific given the evidence above is simply bioinformatic alignments. Following this logic, I would say the more general glycosylase term is preferable. On the other hand, of all the glycosylases, one might argue that the most likely activity for a phage enzyme is as a DNA glycosylase. All of the DNA glycosylases are of course N-glycosylases (i.e. all 4 bases connect to the ribose through a nitrogen). I think the evidence for this phage protein family to be an enzyme is about as good as you can get with just bioinformatic alignment so adding something is probably appropriate, the question is how specific do we want to go, the generalized glycosylase or the more specific N-glycosylase.

Link to this post | posted 09 Nov, 2017 20:42
cdshaffer	What is the recommended protocol for suggesting updates to the newly posted list? For now I will just suggest here that we add "dNMP kinase" to the "do NOT Use" column for the written out entry "deoxynucleoside monophosphate kinase".