Link to this post | posted 08 Feb, 2024 03:25

uOttawaPHAGE

Hi Debbie The question and the evidence has been generated by a student in my course. I think the second gene (25,700 to 26,641) should be annotated as an exonuclease. Clear coding potential, strong functional hits, annotated in all comparators, most used start site that covers all coding potential. I'm not sure what to do for the first gene. Either: 1) Annotate the gene (25,195 to 26,052) as a membrane protein, respect the coding potential and allow a 353 overlap. Two other phages annotate this gene, but as I mentioned above the encoded genes are shorter. No hits on Blastp or HHpred to mention. 2) Leave a gap before the exonuclease and call nothing. I'm skeptical the auto-annotated bottom strand calls are real: only very weak GenemarkS coding potential (dotted red lines)and no matches on HHpred or Blastp. The coding potential is much higher on the top strand. 3) I suppose a third option would be a translational frameshift, though we'd obviously need experimental evidence to make that call. I didn't see any evidence for a slippery site near the transition in coding potential between the frames. I think the decision hinges on whether a 353bp overlap would be acceptable. It severely violates a Guiding Principle. I haven't annotated enough genomes to have a sense what the correct decision is. thanks! Adam

Link to this post | posted 07 Feb, 2024 22:08

uOttawaPHAGE

Hi Debbie Continuing this thread with a different, but related question about Superstar. The genes in question are between 25,200 and 27,000. The auto-annotation added two bottom strand genes, but we see stronger coding potential on the top strand, and we think we should call the gene (a membrane protein with no significant HHpred hits) from 25,195 to 26,052. However, this gene has a very large overlap with the next gene (an exonuclease). To preserve coding potential, use the best RBS scores and match the Glimmer/Genemark calls, the best call for the second gene is 25,700 to 26,641. This would create a 353 bp overlap between the two genes. A related phage, Caelum in the attached document, also has a gene in the same pham on the top strand (gp31 in the attached phamerator map), but with a smaller overlap (~50bp). Would a 353bp overlap be acceptable? Or does the large overlap of the gene at 25,195 to 26,052 in Superstar suggest that the similar gene in Caelum (and Issmi) might have been incorrectly called, and all three should be removed? This would leave a gap and unassigned coding potential. We can move the start site of the second gene, but the start at 25,700 is the most frequently called start and creates a gene in Superstar of similar size to those in related phages. thanks! Adam 360Kb

Link to this post | posted 07 Feb, 2024 21:17

uOttawaPHAGE

Hi Debbie Starting this thread up again with a question from a student, Ashlyn: For draft phage Superstar (BD2), the gene at the coordinates 31,549 to 31,764 (gene 47 on Phamerator) is currently being called a DNA binding protein. However, HHPRED shows high coverage and probability for a helix-turn-helix DNA binding protein. We would like to be as specific as possible for this call. According to the SEA-PHAGES FUNCTIONAL ASSIGNMENTS sheet, the alignments of the protein hitting HTHs must hit 2-3 alpha helices in the sequence separated by small spacer (turn) regions of 3-4 amino acids. However, the alignments I found are showing a spacer region of 6 amino acids between the 2nd and third helix. Is it acceptable to call this as a HTH, or should we simply call it a DNA binding protein? Attached are the top HHpred hits and the sequence comparison for the top hit. thanks Adam 150Kb

Link to this post | posted 07 Feb, 2024 13:04
uOttawaPHAGE	Hi Debbie Ok, this was a misunderstanding on my part. I thought we needed to identify a slippery sequence similar to those that have been defined previously (i.e. on the table). CCCTTTTT definitely works as an XXXYYYZ sequence, and is in the correct position for a -1 frameshift. PF to PF referred to the slip as coding proline-phenylalanine to proline-phenylalanine. thanks Adam

Link to this post | posted 07 Feb, 2024 07:04

uOttawaPHAGE

hi We are annotating phage Aikyam. We noted that phages in this cluster with the same TAC pham, phage Isolde, for example, call the programmed frameshift using a slippery sequence CCCTTTTT (a PF to PF -1 slip). CCCTTTTT doesn't appear on any list of validated slippery sequences that I could find, so I wasn't sure why this site has been called. It certainly works as a slippery sequence, but is there any evidence to support this call? Other AY phages, like EvePickles, use CCCAAAAA (PK to PK; a different Pham), which does appear on Sup Table 1 from Xu et al. thanks Adam

Link to this post | posted 02 Feb, 2024 17:30

uOttawaPHAGE

Hi Debbie The student is carefully annotating this region, and I understand the functional information could influence the start site call, but what if these were two NKFs? I'm interested in the more generic case because it would change the way I think about the annotation guiding principles regarding overlap. If they were NKFs, would I make the same call and use the 44271 start, accepting the 158bp overlap? Based on the coding potential, I think I would want to. Even though Issmi has dissimilar nucleotide sequence, the GeneMark files looks quite similar for all three phages: good coding potential but a lot of overlap. I probably would have included the gene and I was wondering if there is a reason it wasn't changed in QC that I'm missing. Based on synteny, I suspect we'll find the same pham in Issmi (though we haven't checked). A Edited 02 Feb, 2024 17:31

Link to this post | posted 02 Feb, 2024 14:24

uOttawaPHAGE

hi Debbie. To not lose coding potential in gp72, the student selected the start site at 44271 (also has better scores/spacer) and this gives a 158bp overlap. It sounds like you are suggesting we use 44145 to minimize overlap, and not worry about cutting off coding potential? Part of why I posted this is because there is a clear difference in the way the other related genomes were annotated - one QCer added a gene, and I'm guessing the other decided the overlap was too big an issue. I didn't get into the functional calls, which are obviously important, but I first wanted to get an opinion on the 158bp overlap. Phamerator map screenshot attached. Diane on top, Superstar_draft middle, Issmi bottom. Adam Edited 02 Feb, 2024 14:26 125Kb

Link to this post | posted 02 Feb, 2024 01:18

uOttawaPHAGE

We are annotating phage Superstar (BD2) and aren't sure if we should call a large overlap (~150bp) between gp73 and gp72 (~44,250), or a large gap (~300bp) by deleting gp73. Some phages (Ismmi) have deleted the gene, others (Diane) have kept it in. Good coding potential in gp73 (and in the two comparators) that ends abruptly where gp72 begins, but no STOP for ~150bp. My bias (unfounded? Maybe based on my R2I session with Sally Molloy?) is to fill the gap, trust the coding potential, and accept the overlap. Or should we delete gp73? The QCers have had different opinions (though the overlaps do seem to vary somewhat - perhaps more evidence for the idea below). One solution is if there is a programmed slip, and this is "called" (the black triangle) in the Genemark files of Diane and Issmi (actually Diane calls slips between the gp74-gp73-gp72!). My student found a potential slippery sequence in the correct location in Superstar, and we may test it in E. coli, but I know without evidence we shouldn't call this. Any advice/opinions? Adam 143Kb

Link to this post | posted 03 Oct, 2023 15:59

uOttawaPHAGE

Hi Bernadine. Based on an email exchange with Kyle (posted below), he thinks his issue was contamination with Geobacillus stearothermophilus, a thermophile that grows at 55-60 degrees and forms spores, so something that could be hard to get rid of. I also learned that these spores are used to test autoclaves and it is ubiquitous in the environment. My current thinking is our autoclave may not be killing every last spore, but we only see this in our top agar. We've increased the cycle time to 55' until we can have the autoclave checked. We wouldn't see problems in our plates/liquid (unless we put them at 55 - we are trying this) because the bacteria won't grow at 30-37. Not sure if you run the spore tests for your autoclave, but you might want to. Kyle is trying to avoid an extended time at 55, which may work as well, but we can't really make that work for our course. Good luck! Adam Kyle's email to me: Happy to talk this out sometime if it’s helpful. I went to reply on the Forum but I need to reset my password and just decided to do this instead. Re-reading the forum entries and both remembering things from our first bout and also probably forgetting some things, I can say that I feel pretty sure of what was the original cause of our problem. We isolated bacteria from the top agar. We sequenced it. It was Geobacillus stearothermophilus. Naturally, as a thermophile this makes sense. But where did it come from? I believe for us the source was our still. We still have an old distilled water still in our prep lab. We have always used it to make media with. For making regular old media to use at 37C, the presence of Geobacillus would never be noticed. But our phage work is the ONLY work with a hold step at 55C, which is just getting a Geobacillus going…. Given that we have sequencing data to show it is definitely this species, I had our lab manager replace the outflow hose from the still and the problem seemed to go away. Now I give this all to you as preface because, lo and behold!, it is BACK, RIGHT NOW. My TAs this semester were IN the course in SP21 when we had all the problems. And they are very very careful. We’ve done the usual stuff, replacing everything, using water from a different system we have upstairs, autoclaving the hell out of everything, before using containers and then again with media in them. We’ve put the TA in disposable sterile 50 ml conicals. We get contamination everywhere. It seems for us like ANY hold step at 55C is just asking for trouble. Vic suggested at first it could just be calcium precipitation and not contamination, but this looks really biological based on how it grows in the bottles. I am at the moment convinced the only thing we can do is basically never hold the TA at 55C except for the shortest period of time possible. So my plan for Monday is this: We made a couple large glass bottles of top agar. All the usual safeguards, autoclaved the hell out of it, and now we have let the TA bottles cool, and have placed them in the fridge to be solid, happy, and definitely not growing thermophiles, over the weekend. On Monday, we will boil the top agar to liquefy it, then let it cool, aliquot into sterile conicals, and place in the 55C bead baths. When the students are done with their Monday TA we will have them dispose of the TA. We will then rinse and repeat for Weds. This seems like vast overkill, but unless we absolutely minimize our TA time in 55C it seems like a continuous problem right now. Sounds crazy, doesn’t it? We also don’t see contamination in our plates OR in broth. Only the TA.

Link to this post | posted 29 Sep, 2023 03:47

uOttawaPHAGE

Hi Kyle. We think we are having an issue similar to what you describe above. We have just tried new agar and it seems to have helped but not completely. Given the contaminant survives the autoclave, it could be in some of the bottles that are filled with top agar, so I suppose it could persist. I’m hopeful this will fix the problem. Two questions: 1. In your thread you mention you were also having issues with phage propagation, which it sounded like you linked to the top agar issue. Did the propagation issue resolve when the top agar issue resolved? I’m assuming yes, but you didn't say it above. We are seeing propagation issues, but only for a subset of phages. The phages were found on new hosts for us, A. glob 2979 and A. sulf, and we aren't sure if this issue could be more common for these hosts, or if it is linked to the top agar. 2. We aren't seeing contamination in our plates, which is confusing to me. We wonder if this could be because of the cycloheximide. Did you ever consider this? This would suggest a fungal contaminant. Adam