Link to this post | posted 27 Apr, 2017 17:05

debbie

Nancy, I am not sure what you are asking. If you look at the phamerator map of the 3 genomes that you mention, it looks like these is a deletion in the G gene of the G/T frameshift in RitaG. That doesn't have anything to do with the frameshift. The deletion occurred and it looks like the phage can get along without that portion. That shouldn't stop you from calling the appropriate frame shift. I have included how I would call the frame shift in the attached document. if you are asking something else, let me know. Edited 27 Apr, 2017 17:05 117Kb 84Kb

Link to this post | posted 11 Apr, 2017 15:14

debbie

Susan, You can do the patch test on L-agar. Sure, why not! I don't understand the without top agar. the patch test is done to visualize phage release from a colony. So you need wild type smeg in the top agar for that detection. You make a companion plate with the patch test, so that your can carry that colony forward (without picking up WT smeg. does that make sense? You can always use a wire loop. Depending on numbers, that is a lot of firing the loop. You can also use sterile toothpicks for some of this testing.

Link to this post | posted 11 Apr, 2017 15:09
debbie	Nick, I will check with Claire as to what setting he uses. then we can see if they "match". i will keep you posted.

Link to this post | posted 29 Mar, 2017 14:43

debbie

Hi Larry, I am sticking to my call for gene 21. Though my data is slim, my rationale is that the TTG start is under-called and if I can pick it, I do. (That would be my bias - remember it is not favored by any of our gene calling programs, so someone has to stick up for it.) In addition, our limited (very limited) mass spec data suggests that the second start is more likely. (Why? Because the mass spec data has the start of the protein at the next amino acid downstream of the second start.) Until experiments are done, these are predictions. In the big scheme of things, I would find either acceptable. -debbie Edited 29 Mar, 2017 14:43

Link to this post | posted 26 Mar, 2017 17:39

debbie

Ann, Sorry for the delay in this response. I am not speaking specifically and to do this properly, i would need the file and supporting materials to truly evaluate. BUT in this case, I would say it is absolutely correct to look at the reverse frame for possible better calls. You may need to go back to Schwabeltier and check out the evidence for calling that gene (simplest to run GeneMark, I think), because you don't want to perpetuate a call that had no evidence in the first place. When you blast this reverse gene at NCBI - does it hit anything outside of our genomes? That would also validate it a bit. What does HHPRed say? Though we don't see it often, it is acceptable for the C' end of a forward and reverse gene to overlap. I just called one in Arthrobacter phage Jasmine (genes 32 & 33) with a whopping overlap. Note that all the genomes in Cluster DN are drafts, so none have been evaluated. You might want to communicate with the others and work on the difficult ones collaboratively. Remember to include this difficult stuff in your cover letter to SMART. Keep up the good work!

Link to this post | posted 26 Mar, 2017 17:06

debbie

Larry, Hi. Your questions are thoughtful and difficult. First, I can' order the tools as you would like me to. each case is solved in context, so i can't prescribe a weight to any individual piece of data. Second, there are 5 Cluster T genomes, only 2 of which are finals. My opinion is that all 5 inform your decision making, so you can't ignore the data just because they are drafts. Soon, we will have to reconcile all of the cluster T phages, but I can' tell if we still have enough data. So, I started to look at gene by gene. I would not worry about which previous call I agree with(when they differ - between the 2 in GenBank). Just make your best call. Your cover sheet to SMART will be lengthy in places where you don't like one of the final 2 choices better than the other. Here is as far as I got so far: gp 21 I would call 19663 gp 26 I would call 23229 gp 30 I would call 26046. It is the only start that captures ALL coding potential. It took some work to throw away the GeneMark call on the opposite strand. Cool. gp 32 I think that I would call 27508. I don’t like any of the choices, but this is the one that captures all coding potential but doesn’t have a shitty SDS. gp 33 This one is hard. I’m still thinking on this. I don’t find that the starerator program is all that helpful. I need to think about this one again. It is the integrase, so we should be able to see the different domains that need to be there…. and it is significantly different from Bxb1's integrase (the one with experimental data to determine the start). gp 34 This one is the immunity repressor. Again, I couldn't resolve this one easily. it might be helpful to look for the attP site around here too. That is all I have for now. i will email Welkin to ask that she chime in. Keep up the good work, debbie

Link to this post | posted 23 Mar, 2017 23:50

debbie

I am going to add to Chris' correct statement. The "functional annotation" does eventually get put in the product field - by SMART. But you are asked to place functions in the notes. There are buttons in DNA Mater that move the contents of the Notes, Functions, and Products fields. We ask for it in the Notes so that as we overwrite the final file, it will format correctly. So your thinking is logical, our protocol is simply a logistics request to minimize the amount of re-formatting done at QC.

Link to this post | posted 13 Mar, 2017 15:39
debbie	Kayla, I use big plates - 150 x 15mm. You can do fewer lysates/plate if you want to use the 100 x 15mm. Whatever you have available is great! Thanks, debbie

Link to this post | posted 03 Mar, 2017 16:54

debbie

Victoria, Mass spec data of a few of these instances shows the protein starting at the amino acid next to the second start. Since it is common that the first methionine gets chopped of in post-translation processing, that NO methionine is present in the mass spec reads, it is likely that the second methionine initiated translation. So in this example, the 4 bp overlap gets 'trumped'. This decision is based on the simplest possible answer. and never forget, things do not have to be simple!

Link to this post | posted 11 Feb, 2017 16:39
debbie	Vassie and Lee, Happy visiting! You can post your results on this forum under the topic "Post Results Here". I also provided a format, so that the results reporting could be consistent. The same procedure is in place at the Xeno Challenge Forum for those results. I am curious to see if these mechanisms work, so by all means, post away! Thanks! Edited 11 Feb, 2017 16:40