SEA-PHAGES | All posts created by c.sunnen

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

Posted in: General Message Board → A Message from David Asai

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.

Posted in: Frameshifts and Introns → Frameshift in DY phage Tarzan (and others)

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 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.

Posted in: Frameshifts and Introns → Frameshift in DY phage Tarzan (and others)

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 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!

Posted in: Cluster DJ Annotation Tips → No coding potential where others call a gene

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)!

Posted in: Choosing Start Sites → Integrase makes a 4bp overlap with immunity repressor?

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

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.

Posted in: Choosing Start Sites → Integrase makes a 4bp overlap with immunity repressor?

Link to this post \| posted 10 Sep, 2022 17:36
c.sunnen	For anyone that may be helped by this, installing that library worked like a charm!

Posted in: DNA Master → DNAMaster BLAST failure

Link to this post \| posted 10 Sep, 2022 16:30
c.sunnen	Hmmmm. I wonder if I'm having this issue. I've tried BLASTing a single gene, and now I get the error "Could not load SSL library." I'll try the fix below, and let everyone know if it works. Nikki hgavin90 DanRussell Hi Katie, I think this error is due to not having a certain library installed on the particular version of Windows that's running. That library wasn't required until after the update earlier this year to use secure NCBI servers. I think I came across that error myself, and in my notes I have that I went to the link below and installed the Visual C++ package there, then restarted and it worked. ~~https://www.microsoft.com/en-us/download/details.aspx?id=5555~~ UPDATE Sep 2021: The link above no longer works, but the file can be found at the link below now. Download it from within Windows and then run it, and make sure you close DNA Master and restart Windows before trying to BLAST again. https://phagesdb.org/static/vcredist_x86.exe Hopefully that helps, –Dan Nevermind – changing secure connection did not work but this installation recommended by Dann has. Woo!

Link to this post | posted 10 Sep, 2022 16:30

c.sunnen

Hmmmm. I wonder if I'm having this issue. I've tried BLASTing a single gene, and now I get the error "Could not load SSL library."

I'll try the fix below, and let everyone know if it works.

Nikki

hgavin90
DanRussell
Hi Katie,

I think this error is due to not having a certain library installed on the particular version of Windows that's running. That library wasn't required until after the update earlier this year to use secure NCBI servers. I think I came across that error myself, and in my notes I have that I went to the link below and installed the Visual C++ package there, then restarted and it worked.

~~https://www.microsoft.com/en-us/download/details.aspx?id=5555~~

UPDATE Sep 2021: The link above no longer works, but the file can be found at the link below now. Download it from within Windows and then run it, and make sure you close DNA Master and restart Windows before trying to BLAST again.

https://phagesdb.org/static/vcredist_x86.exe

Hopefully that helps,
–Dan

Nevermind – changing secure connection did not work but this installation recommended by Dann has. Woo!

Posted in: DNA Master → DNAMaster BLAST failure

Link to this post \| posted 10 Sep, 2022 12:52
c.sunnen	Veronique, Did this ever fix for you? It looks like I might be having the same issue. It's almost as if DNA Master just didn't even start sending queries. It stayed at 0% overnight with no progress. Is this what happened for you? Nikki

Posted in: DNA Master → DNAMaster BLAST failure

Link to this post \| posted 03 Jun, 2022 21:08
c.sunnen	Thank-you, Christian! I see that now in my flat file. It's weird though, because the DNA Master file has "tRNA-Ile2(cat)" in the product field (and properly in the Documentation), so I don't know why it's getting changed in the flat file. Maybe I can just edit the flat file directly and resubmit; the sub file appears accurate. Thank-you! Nikki

Posted in: tRNAs → Met vs Ile2

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