Link to this post | posted 07 Aug, 2022 18:04

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More info added in bold Hi, I have a (circular) genome that was assembled at NCSU and its closest hit is a phage that was annotated in 2010 at Wellcome Trust Sanger back in 2010 (NC_015296.1). So, no UPitt assembly QC on either, I'm afraid. Hopefully, someone will help me wrap my head around this. The strand is completely opposite between the two and there is a relative inversion in the middle. That is, all my forward genes are listed as complement in the other phage and vice versa. But, (with gp210 approximately the ends of both) the gp numbers are partially inverted gp1+strand=gp64-strand, gp2+strand=63-strand….. gp61-=gp1+; gp 62-=gp210+, gp 63=209….etc). So, it is like there are two giant crossovers if you map them in a phamerator fashion. I know that BLASTN searches also the reverse complement, but all of these genes are listed as Q1:S1 essentially, not reverse. So, I think even if I assume that my genome should have gp 61 as gp1, there is still an inversion relative to each other. Otherwise, I would have gp1=gp210, gp2=gp209…. Finally, there are other phages (published more recently but less sequence conservation) that have the same strand but whose halves are flipped relative to mine. gp1+=gp142+, gp27-=gp168-, gp32-=gp172- So, my current hypothesis is that phage A may be in GenBank as reverse complement but that doesn't matter. Coding is strand agnostic, so I think if I had gp1=gp210, gp2=gp209…. But, we have about 80% NKF and so large-scale functional synteny is a challenge but tapemeasure protein appears to be after the transition from forward to reverse around our gp 30. So it is early in the genome with our gp1 but not in the forward direction, and a couple of tail fiber proteins are "after" tapemeasure as reverse genes. So that would indicate that our assembly is maybe entirely reverse complement compared to the consensus setup? Q1: Is it possible that my assembly should be inverted in some way? "Complementing the contig" for the whole genome? (Even if I were to base it on the reverse complement, it would still have an inversion compared to the other.) Q2: Or, should I start with gp1 in the middle and flip the halves? Thanks.

Link to this post | posted 07 Aug, 2022 17:41

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Hi, I have a (circular) genome that was assembled at NCSU and its closest hit is a phage that was annotated in 2010 at Wellcome Trust Sanger back in 2010 (NC_015296.1). So, no UPitt assembly QC on either, I'm afraid. Hopefully, someone will help me wrap my head around this. The strand is completely opposite between the two and there is a relative inversion in the middle. That is, all my forward genes are listed as complement in the other phage and vice versa. But, (with gp210 approximately the ends of both) the gp numbers are partially inverted gp1+strand=gp64-strand, gp2+strand=63-strand….. gp61-=gp1+; gp 62-=gp210+, gp 63=209….etc). So, it is like there are two giant crossovers if you map them in a phamerator fashion. I know that BLASTN searches also the reverse complement, but all of these genes are listed as Q1:S1 essentially, not reverse. So, I think even if I assume that my genome should have gp 61 as gp1, there is still an inversion relative to each other. Otherwise, I would have gp1=gp210, gp2=gp209…. Finally, there are other phages (published more recently but less sequence conservation) that have the same strand but whose halves are flipped relative to mine. gp1+=gp142+, gp27-=gp168-, gp32-=gp172- So, my current hypothesis is that phage A may be in GenBank as reverse complement but that doesn't matter. Coding is strand agnostic, so I think if I had gp1=gp210, gp2=gp209…. for the whole genome it would not matter, right? Q1: Is it possible that my assembly should be inverted in some way? "Complementing the contig" for the whole genome? Even if I were to base it on the reverse complement, it would still have an inversion compared to the other. Q2: Or, should I start with gp1 in the middle and flip the halves? Q3: Alternatively, am I thinking about this whole thing stupidly and there really is no consensus on which strand or where to start gp1? Thanks.

Link to this post | posted 26 May, 2022 14:38

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I never got a response from MPI. I tried it with and without logging in and it didn't seem to matter. Instead of the usual copying over the previous input, I started hitting HHPRED again to reload the search tool and not using the job ID and it seems to be ok now. (it is saving the three databases but I have to keep track of which GP). (It is also possible that it was a transient server or traffic problem on their end). But it's working now! Edited 26 May, 2022 15:05

Link to this post | posted 19 May, 2022 17:02
stpage	They never emailed me back but we seem to be ok if we are rather gentle with HHPRED. Specifically, don't: -copy over a previous input and resubmit under a new ID -launch multiple HHPRed searches simultaneously under the same browser Sometimes, it seems to help to erase the previous job before entering a new job and launching. It's nice that I can still find inventive ways to break things.

Link to this post | posted 11 May, 2022 12:41

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Um, has anyone ever gotten kicked out of HHPRED? My student and I were launching multiple HHPRED jobs (3-4) in multiple tabs and now both of our IPs have been blocked apparently. I can search on a different laptop but when we search on those two laptops, we get "ERROR!". We tried to clear cookies and restart and nope, HHPRED still throws the error, so I think it might be IP-based. I get the error whether I am logged into an HHPRED account or not. If we are the first, um, well, word to the wise. Edited 11 May, 2022 12:43

Link to this post | posted 14 Mar, 2022 14:25
stpage	Hi, we had this discussion when we annotated MarioKart a little over a year ago. I am not sure that there is not a frameshift but just felt like there wasn't enough evidence to call it. Nhagos and Sours have 4 Gs around the end of gene 21 that is the region identified as the slippery sequence. Does yours have Pham 113375 or 115039? It might be worth tracking it by Pham rather than by cluster.

Link to this post | posted 15 Nov, 2021 17:17
stpage	fogartym1 Thanks Vic! We will try it out and report back on how it goes. Marie Hi, did you ever have any luck with this? I have a student who has apparently lost their phage that they isolated a couple of weeks ago. We'd like to take his old plaques and PB and amplify them back up. Thanks.

Link to this post | posted 27 Oct, 2021 23:34
stpage	Thanks, Vic. That is helpful. We were losing some DNA and weren't sure if we were getting capsid denaturation from the proteinase K. Different protocols seem to have wildly different proteinase K conditions.

Link to this post | posted 27 Oct, 2021 19:27

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Hi, the phage discovery has RNase/DNase/ProteinaseK treatment followed by CleanupResin (guanidinium thiocyanate) to denature the capsid. Some protocols seem to use proteinaseK to denature the capsid. (https://www.mdpi.com/2409-9279/1/3/27/pdf-vor) and some seem intermediate (https://pubmed.ncbi.nlm.nih.gov/29417429/). Is it very phage dependent? With new hosts do we need to optimize the proteinaseK treatment??

Link to this post | posted 26 Feb, 2021 18:57
stpage	There are 6 B1 phages in phagesdb calling the holin.