Link to this post | posted 19 Mar, 2021 16:52

joycestamm

Thanks all, for the discussion! I walked through this with my students in class yesterday, having them think first about why the programmed translational frameshift wasn't called in any of the cluster members, and then coming up with the evidence that we'd need to annotate the frameshift. Then each group examined part of the evidence (we didn't have time for everyone to do all of it), and we came back together to discuss. They were pretty convinced about the CCCAAAT pattern. Then I showed them the forum discussion, and Chris's comment got posted as they looked at it, which was super cool. I thought it was a great example of how science is done, and the students were really excited to be part of it. I'll email you, Chris, to get the sequences/alignment. Thanks again!

Link to this post | posted 18 Mar, 2021 01:32

joycestamm

So if I had posted this a day or two ago, our poster abstract might have looked quite a bit different… Would it be ok for us to try to include all this analysis in our poster? Maybe we'd get more insight, or at the very least, this looks like a good learning opportunity for my students. As for what I think - I don't have anywhere close to everyone else's experience with phage genome annotation, but it seems to me that the high level of conservation in the region between the first chaperone and the "second" chaperone is another piece of evidence suggesting that the region codes for protein. But I appreciate that there is an issue with precisely calling the slippage position. But the sequence conservation within the pham does seem to support Debbie's CCCAAAT suggestion. So I *want* to call the slippery sequence there, but I also agree that we'd be making an educated guess. It would be nice to come to some kind of conclusion - we're slated to annotate a couple of the BE2 phages shown in Chris's figure for the second half of the semester!

Link to this post | posted 17 Mar, 2021 21:19
joycestamm	So is the idea that if there isn't a slippery sequence that matches one of the ones that have been experimentally determined, then we assume that there isn't a programmed frameshift? Should we annotate the small, 29 aa coding sequence as a protein then? That's smaller than given in the guiding principles.

Link to this post | posted 17 Mar, 2021 19:32

joycestamm

I have a question about the tail assembly chaperone protein in cluster BK1. We are annotating the Emma1919 genome. Looking at all non-draft BK1 genomes in Phamerator, none of them show a tail assembly chaperone with a programmed translational frameshift. Instead, they show two consecutive tail assembly chaperones: the first one belonging to pham 5495 and the second belonging to either pham 22821 or pham 37280. I ran an NCBI blastx between the Emma1919 genome and the second tail assembly chaperone (from pham 37280) in Gilson (Gilson_39), which is the most similar genome to Emma1919, and found an almost perfect match between Gilson and Emma1919, except at the start codon (Emma1919 has a GTG so has a V instead of M). But the protein is only 29 amino acids long. Is this very short second protein legitimate? Or is it more likely that there is a translational frameshift in here that was previously missed? If so, how can we find the slippery sequence? The attached picture shows the six-frame translation of the region of interest. The first tail assembly chaperone (pham 5495) is highlighted in yellow, and the pham 37280 protein is highlighted in green. The region in blue in between the two ORFs doesn't contain any stop codons, which suggests to me that there might be a frameshift somewhere in the nucleotide sequence shown in red. A few more data points: GeneMark shows atypical coding potential in that whole blue/green region. The entire region (bp 22601-23300) is highly conserved with Gilson, with only 6 bases different between the two. The DNA sequence in the region where the frameshift has to happen doesn't contain a sequence that matches the known slippery sequences shown in the Bioinformatics Guide. Edited 17 Mar, 2021 20:04 29Kb

Link to this post | posted 09 Jan, 2018 23:20

joycestamm

More information: I have tried opening 3 different C1 cluster phage files, and gotten the access violation message every time. I have tried opening 3 different non-C1 cluster phage files, and it's worked every time. I uninstalled wine and DNA master, reinstalled, updated, and tried again. Still no dice. I've tried opening the C1 cluster file on 3 different Macs, and failed each time. But I was able to open the file and create the /dnam file on the one PC that I have access to. So it appears to be a Mac-specific, longer sequence-specifc error?

Link to this post | posted 09 Jan, 2018 18:44

joycestamm

The error is happening much earlier, and is inconsistent. I have gotten it when I'm just trying to open the fasta file (so at the Open FastA multiple sequence file) point. Sometimes I manage to open the file, but when I try to Export it to "Create sequence from this entry only" I get the error message. Then DNA master crashes and I often have to force-quit. I'm working on a Mac, if that matters. And this problem is happening on both the Macs in my office and at home, both of which worked last year.

Link to this post | posted 09 Jan, 2018 03:45
joycestamm	I've been trying to open my new phage sequences in DNA Master. I have three sequences - one of them opened normally and I was able to save a DNAM file. But I can't get the other two to work - I keep getting an error message that says Access violation at address … (see the attachment). Does anyone know what this means, and more importantly, how to fix it? 19Kb

Link to this post | posted 08 Jul, 2017 20:14
joycestamm	Thanks, Steve and Lee for your help! To check if I'm understanding you correctly - I would make the spore suspensions in lieu of 'regular' freezer stocks, correct? Edited 08 Jul, 2017 20:15

Link to this post | posted 03 Jul, 2017 15:53

joycestamm

Apologies for the newbie question. I'm hoping to try to use Streptomyces as a new host this fall, and would like to do a run-through with my summer research students. I've read through the updated versions of the instructor guide and the Streptomyces specific phage discovery guide, but haven't found the answer - how do I make a spore suspension? Also, I read the previous thread on this forum with interest. Do you have an opinion as to whether I should use the spread technique or top agar? The phage discovery guide says top agar, because a germination spore preparation is not practical for large scale use, but have you found that to be true? Thanks in advance for your help.

Link to this post | posted 18 Jan, 2017 03:29

joycestamm

One of my students is having problems installing DNA Master on her Windows 10 computer. When she tries to run the program, she always gets a screen where she can either repair, modify, or remove the program. She has tried selecting either repair or modify, but it doesn't seem to do anything, and she can't get to the program. She took her computer to our computer support people, who simply said that her computer isn't compatible with the program, but that doesn't make sense to me. Any suggestions?