Link to this post | posted 04 May, 2024 13:29
c.sunnen	Thanks, Debbie. Will check back in later, before we send it in.

Link to this post | posted 03 May, 2024 17:55

c.sunnen

The paper you linked has a nice RNAseq map of Fruitloop in Figure 4, in addition to the mass spec data in Table 1. Yes, that's very helpful! Fruitloop does have 95% sequence similarity in this region to PhesterPhotato, and Fruitloop doesn't call either of these genes (46 and 47 in PhesterPhotato). When I look at GM-S coding potential from Fruitloop in this area, there is a little CP in the forward direction in the gap between 43 and 44 (between 45 and 48 in PhesterPhotato), but it's only atypical, and not as strong as it is in PhesterPhotato. The RNA seq from Fruitloop makes it look like MAYBE there's a reverse gene in there that gets expressed during lysogeny, but it's hard to see, and definitely there's not a forward gene expressed. The mass spec doesn't show these genes being made (nor the flanking reverse genes, either though, including the immunity repressor), but I guess that's still evidence that neither of these is real. I'll delete both 46 and 47 from PhesterPhotato, so that it matches with Fruitloop. Thank-you!

Link to this post | posted 03 May, 2024 17:55

c.sunnen

The paper you linked has a nice RNAseq map of Fruitloop in Figure 4, in addition to the mass spec data in Table 1. Yes, that's very helpful! Fruitloop does have 95% sequence similarity in this region to PhesterPhotato, and Fruitloop doesn't call either of these genes (46 and 47 in PhesterPhotato). When I look at GM-S coding potential from Fruitloop in this area, there is a little CP in the forward direction in the gap between 43 and 44 (between 45 and 48 in PhesterPhotato), but it's only atypical, and not as strong as it is in PhesterPhotato. The RNA seq from Fruitloop makes it look like MAYBE there's a reverse gene in there that gets expressed during lysogeny, but it's hard to see, and definitely there's not a forward gene expressed. The mass spec doesn't show these genes being made (nor the flanking reverse genes, either though, including the immunity repressor), but I guess that's still evidence that neither of these is real. I'll delete both 46 and 47 from PhesterPhotato, so that it matches with Fruitloop. Thank-you!

Link to this post | posted 02 May, 2024 21:23

c.sunnen

Hi! We are annotating PhesterPhotato, an F1 cluster phage, and it's got some extra weirdness, even for an F1! Please see the attached Phamerator + Genemark output picture. There's a forward gene (47) in the middle of a small group of reverse genes. It overlaps. But there may be a reverse gene instead (46). There's evidence for both, though better CP for the forward (47) than the reverse. Some other F1s call the forward (12 total members in pham), some call the reverse gene (again, 12 total members). If I call the reverse, in order to capture the CP, I'd have to create an overlap with gene #48, which no one does, so I'm feeling like this tiny reverse gene isn't real. But why would there be a forward in the middle of these reverse? Creating an overlap? Most other phages don't call either, and just leave a big (300bp) gap. Please help. It's in PECAAN if you want to look at it there. No evidence for function for either 46 or 47, but 48 is likely the immunity repressor. Thanks! Nikki & the SJU team 1.89Mb

Link to this post | posted 12 Jan, 2024 20:59
c.sunnen	Thank-you, David, for all that you have given to us, to the program, and to the students. Congratulations on your much-earned retirement! You will be missed, but your legacy will live on. Congratulations! Nikki Sunnen Associate Prof of Practice Saint Joseph's University (and formerly University of the Sciences) csunnen@sju.edu

Link to this post | posted 17 Mar, 2023 12:49
c.sunnen	Thanks for that, Debbie! I often wonder what exactly makes the sequence "slippery" other than the obvious repeat of bases, but it's nice to know that as long as the amino acid sequence comes out right, we don't have to worry too much about which coordinate/base we annotate as being repeated.

Link to this post | posted 16 Mar, 2023 18:27

c.sunnen

I am QC-ing the frameshift in DY cluster phage Tarzan. It is a GN to GK, -1 frameshift with a slippery sequence of GGG.GGA.AAC, where it appears that the first A gets counted twice. This is most similar to the GN to GK slip seen as #15 in the table within the Bioinformatics guide (GGGAAAT). However, 2 of the 3 annotated phages (Jojo24 and Reyja) annotate the slip at the third G instead, making the slip actually a GG to GG. I lean towards the GN to GK, since it's more similar to others where it slips at the first A, but perhaps there's something I don't know? Which one is it? Screenshot from Tarzan attached. 1.22Mb

Link to this post | posted 13 Mar, 2023 15:58

c.sunnen

Hi Erin! It looks like inserting a gene here, like your close relatives did (and deleting the reverse that was called) will probably give you nice 1 or 4bp overlaps, with both the upstream and downstream genes. Check to see; if so, this is really good evidence that you have a gene there. And if you BLAST this ORF, do you get 1:1 alignments with your close relatives? Also good evidence. It looks like the frame that you DO have coding potential in (that would create the 69bp overlap) is also present in your relatives. I think of this non-specific coding potential as "bleed-over" from other frames. Sometimes it's real, sometimes it's not, sometimes it's "misplaced" in the incorrect frame (this is not particularly scientific, just my interpretation when I see these things). But it does seem to be consistent between the genomes you shared, so there's probably something in that region, but maybe not in that frame. Remember also that the GM outputs are generated anew each time they're run, so it's conceivable that you would see coding potential in that region, in the appropriate frame, in another iteration of running Genemark. You may also want to check to see what the GM output looks like when you run it trained on the host (I assume the one you're showing is trained on Self). Sometimes you'll get different information from each one. Generally, even if there's no CP for your phage, if close relatives have called a gene there and I get 1:1 hits, I'd insert the gene. CP and/or 1:1 Blast hits are good enough for me. I'd rather call a gene that isn't there than NOT call one that is!

Link to this post | posted 23 Nov, 2022 16:17
c.sunnen	Thanks, Debbie! I really just needed someone else to confirm this for me (or to tell me favored overlaps are a thing with integrases sometimes). Abysmal RBS it is (and yes, it is the best conserved of the options, even though it's not always the one called)!

Link to this post | posted 23 Nov, 2022 14:20

c.sunnen

Hi all, We are annotating DY cluster phage Tarzan; there are only 5 members of this cluster, of which only 3 (Santhid, JoJo24, and Reyja) have been manually annotated. I am looking at the tyrosine integrase (gene 31 in Tarzan), and having trouble weighing the evidence for the best start. In short, I can call a favored 4bp overlap with the immunity repressor (gene 32) OR I can leave a gap and choose the start with the abysmal RBS (which is know is common for integrase genes). Are favorable overlaps also seen between integrase and immunity repressor if they're right next to each other? I seem to recall that integrase usually has an upstream gap… Some details: Starterator is not very helpful. Though there are 45 members of the pham, there is no real consensus; the most annotated start is only called 9/42 times (and only present in 22% of genes), and none of the phages in this cluster have it anyway. Within the cluster - which do look similar, but not identical - they all call different starts. However, while none of them called it, all 3 (Santhid, JoJo24, and Reyja) could have called a start that would've given a favorable 4bp or 8bp overlap with the immunity repressor. I've attached a summary of start options for each of these 4 genes (including Tarzan). Any insight would be appreciated. I'm leaning toward the 4bp overlap, but something tells me that's a "no no" for an integrase. 16Kb