Link to this post | posted 27 Feb, 2016 17:16
cdshaffer	David, My bad, I mis-clicked on the permissions and only shared with other WashU people, so I think the "fee" to get access would probably be more properly termed "tuition". I don't think we want to wait that long so I changed permissions, RcigaStruga should now be available from the link above to anyone. Here are the links to the other reports, easy peasy. Christian, Huntingdon, Canowicakte.

Link to this post | posted 26 Feb, 2016 20:31

cdshaffer

Don't worry too much about time. It takes me about 3 minutes to start the run (then the computer works on its own) and about 5 minutes to post the results if they work. If they don't work and still crash starterator then I really do want to document that so we have as complete a list as possible of potential bugs. I got Suppi and RcigaStruga to work just fine with my updated code. I then tried with the original version of Starterator without my code updates and Suppi worked there as well. So now I wonder if this is something specific with your machine. See the sticky first message in the starterator section on common troubleshooting, you should in particular try to delete all the cached Intermediate files (item 2) as that can cause problems if you try to run starterator on phage and the phage get updates in the database. If your phage are still crashing I wonder if you have a corrupted database. You might want to try opening phamerator and doing a force database update. Also try a couple of machines see if you get crashes on only one or all of them. If all those don't help let me know, I can post more reports. easy enough to do over the weekend.

Link to this post | posted 26 Feb, 2016 16:22

cdshaffer

Nancy, It looks like Starterator was crashing on LouisV14 for an already solved problem, as my local version of starterator with my added code worked just fine and I was able to make a full report. You can download the full report here. David, If I remember correctly the individual gene routine still needs a lot work Starterator crashes on many negative strand genes and if I remember correctly it also does not work well if you give it an orpham. Like you, my early experience in class was very sporadic, some genes worked others did not. Consequently I abandoned that routine in class pretty quickly and worked on getting the whole phage report working since that produced a PDF that could be used for the remainder of the semester. I have not worked on any bugs in the single gene routines at all really, instead, putting what time I could on the bugs that cause the whole phage report to crash. Can you send me the names of one (or two) phage and which pham was being worked on when starterator crashed. I am more interested in which phage cause crashes then doing a whole phamerated phage report as that (I think) is the most important routine to debug but any well documented reports of crashes are always welcome. As long as I get get them to repeatedly crash I can put them on the "to do" list of bugs to investigate and fix. Edited 26 Feb, 2016 18:33

Link to this post | posted 25 Feb, 2016 16:03

cdshaffer

I had the same issue with some of our recently isolated Streptomyces phage. Several of them were so novel across the whole genome based on BLASTn that I had to go the "find the terminase" route. Looking at some of the previous Streptomyces phage the terminase is not always the very left most gene, so I matched those phage and I ended up moving to the left several genes to find a "clearing" in the coding potential and then pick base 1 somewhere that would avoid dividing any long ORFs. You can look at phage Yara_draft in phamerator as an example. A few of our phage were similar to Yara so we used that as a guide. In Yara the similarity to terminase starts at around 2.5 kb and is the 8th gene called by DNA Master auto-annotation.

Link to this post | posted 24 Feb, 2016 21:16

cdshaffer

The pdf library in phamerator is not very good so if you save a map from phamerator directly to PDF and try to print in large form it does not work well. Here is the recommended protocol I give my students: 1. Open phamerator and create your map. 2. Edit the view menu to set what is shown and not shown to suit your purpose 3. Save as a SVG from phamerator. 4. Transfer the file as needed to a computer which has Inkscape installed. (Inkscpe is a free to download graphics program that works kind of like Illustrator. There are versions for all platforms) 5. Open the file in Inkscape and use save as… to save in PDF format 6. Open the PDF version of the file in whatever program you are using to create your poster 7. Typically students are using Powerpoint to create a giant slide, in which case I tell them to use the Insert menu -> photo -> picture from file to select the PDF. Then resize the map to fit the size of the poster.

Link to this post | posted 24 Feb, 2016 18:30

cdshaffer

Just as a check I wrote a quick biopython script to ran a blast search using the qblast server at NCBI and it worked, at nearly the same time I tried to submit a search from DNA Master and I too am getting the same error. This would suggest that DNA Master is the issue and it is not a general problem with the NCBI qblast service. So it is possible DNA Master (from all of us) has submitted too many requests in too short a time and NCBI has blocked DNA Master (i.e. all copies everywhere) from submitting any more searches. If this is indeed the case and if I understand NCBI policy in this regard Dr. Lawrence needs to get in touch with NCBI and get DNA Master on some kind of a whitelist so we don't get blocked. Dr. Lawrence should know more about these NCBI policies. P.S. HAHA I only have to wait 58 million seconds, apparently NCBI likes me more than you Edited 24 Feb, 2016 20:30

Link to this post | posted 24 Feb, 2016 17:46

cdshaffer

OK thanks. Looking more closely we have now found at least several tRNA's where there are NO bases between the end of the tRNA and the first base of the auto-annotation. Splicing of the RNA to excise the tRNA would thus produce a leaderless message. I have been talking to our local faculty who works almost exclusively with Streptomyces (the phage host in this case) and apparently in some of these strains leaderless messages are not that uncommon, so I think we will go ahead and annotate both no matter how close as along as they don't overlap. I tend to agree with the general annotation policy that it is better to annotate a false positive than a false negative; so when things are truly ambiguous over-predict don't under-predict. He also raised the possibility that this is an intentional organization that could be used as some sort of regulation of expression, by splicing out an overlapping tRNA you effectively kill the protein encoded gene, resulting in less protein production than if the tRNA wasn't there. I don't know if this has ever been described before but would be very cool if true.

Link to this post | posted 24 Feb, 2016 17:42
cdshaffer	Yes the intermittent nature is very curious. Steve has updated the bzr archive with Actino_draft as the default. Please let us know if that seems to fix the problem. If not, then I agree with you, may take some serious doing to track down the issue, so if anyone else is having these issues please post here. Edited 24 Feb, 2016 17:43

Link to this post | posted 19 Feb, 2016 21:27

cdshaffer

Good news, my updated code worked for this one. You can download the complete file here. Wow! over 550 pages, that's some big phams. Sorry it took me so long, too much grading to stop by SEA Phages the last couple days. If anyone is in a hurry feel free to send me an email note that you posted a request and I will get on it, it really doesn't take long to set up the run and then the computer does all the work so don't hesitate to ask! Edited 19 Feb, 2016 21:30

Link to this post | posted 18 Feb, 2016 16:58

cdshaffer

OK, general question. We have a new phage with lots of tRNA genes packed interdigitated with glimmer/Gene protein predictions. There are long spaces between the tRNA genes so there is certainly room for a protein gene but some of the glimmer and genemark predictions come pretty close to the aragorn hits. How much space should there be between these features? Do we need to leave room for a promoter anytime we switch from a tRNA gene to a protein gene or vice versa? Should I tell the students to leave at least 25-50 bp needed for the promoter? I will have students look at the protein vs. tRNA distribution in the host bacteria but wanted to check if there is some kind of general rule to maintain consistency across all SEA phages annotations. Edited 18 Feb, 2016 17:34